Anti-CD22 Antibodies and Immunoconjugates and Methods of Use

ABSTRACT

Anti-CD22 antibodies and immunoconjugates thereof are provided. Methods of using anti-CD22 antibodies and immunoconjugates thereof are provided.

This is a divisional application filed under 37 CFR Section 1.53(b)claiming priority to U.S. application Ser. No. 11/754,899 filed May 29,2007, currently pending, which claims priority under 35 USC Section119(e) to U.S. Provisional Application Ser. No. 60/809,328, filed 30 May2006, now expired, 60/908,941, filed 29 Mar. 2007, now expired, and60/911,829, filed 13 Apr. 2007, now expired, the entire contents ofwhich applications are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to anti-CD22 antibodies andimmunconjugates thereof. The invention further relates to methods ofusing anti-CD22 antibodies and immunconjugates thereof.

BACKGROUND

Lymphocytes are one of many types of white blood cells produced in thebone marrow during the process of hematopoiesis. There are two majorpopulations of lymphocytes: B lymphocytes (B cells) and T lymphocytes (Tcells). The lymphocytes of particular interest herein are B cells.

B cells mature within the bone marrow and leave the marrow expressing anantigen-binding antibody on their cell surface. When a naive B cellfirst encounters the antigen for which its membrane-bound antibody isspecific, the cell begins to divide rapidly and its progenydifferentiate into memory B cells and effector cells called “plasmacells.” Memory B cells have a longer life span and continue to expressmembrane-bound antibody with the same specificity as the original parentcell. Plasma cells do not produce membrane-bound antibody but insteadproduce the antibody in a form that can be secreted. Secreted antibodiesare the major effector molecule of humoral immunity.

B cell-related disorders include, but are not limited to, malignantlymphoma (Non-Hodgkin's Lymphoma, NHL), multiple myeloma, and chroniclymphocytic leukemia (CLL, B cell leukemia (CD5+B lymphocytes).Non-Hodgkin's lymphomas (NHLs), a heterogeneous group of cancersprincipally arising from B lymphocytes, represent approximately 4% ofall newly diagnosed cancers (Jemal, A. et al., CA-Cancer J Clin, 52:23-47, (2002)). Aggressive NHL comprises approximately 30-40% of adultNHL (Harris, N. L. et al., Hematol. J. 1:53-66 (2001)) and includesdiffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL),peripheral T-cell lymphoma, and anaplastic large cell lymphoma.Frontline combination chemotherapy cures less than half of the patientswith aggressive NHL, and most patients eventually succumb to theirdisease (Fisher, R. I. Semin. Oncol. 27(suppl 12): 2-8 (2000)).

B cell-related disorders also include autoimmune diseases. Autoimmunediseases remain clinically important diseases in humans. As the nameimplies, autoimmune diseases act through the body's own immune system.While the pathological mechanisms differ among individual types ofautoimmune diseases, one general mechanism involves the binding ofcertain antibodies (referred to herein as self-reactive antibodies orautoantibodies) to the body's endogenous proteins. Physicians andscientists have identified more than 70 clinically distinct autoimmunediseases, including rheumatoid arthritis, multiple sclerosis,vasculitis, immune-mediated diabetes, and lupus such as systemic lupuserythematosus. While many autoimmune diseases are rare—affecting fewerthan 200,000 individuals—collectively, these diseases afflict millionsof Americans, an estimated five percent of the population, with womendisproportionately affected by most diseases. The chronic nature ofthese diseases leads to an immense social and financial burden.

Cytotoxic agents which target B cell surface antigens are an importantfocus of B cell-related cancer therapies. One such B cell surfaceantigen is CD20. Rituximab (Rituxan; Genentech, Inc. (South SanFrancisco, Calif.) and IDEC Pharmaceutical Corp. (San Diego, Calif.)), achimeric (mouse/human) anti-CD20 monoclonal antibody, was the firsttherapeutic antibody approved by the United States Food and DrugAdministration for treatment of relapsed or refractory low-grade orfollicular NHL (Leonard, J. P. et al., Clin. Canc. Res. 10:5327-5334(2004)).

Other B-cell antigens, such as CD19, CD22, and CD52, represent targetsof therapeutic potential for treatment of lymphoma (Grillo-Lopez A. J.et al., Curr Pharm Biotechnol, 2:301-11, (2001)). CD22 is a 135-kDaB-cell-restricted sialoglycoprotein expressed on the B-cell surface onlyat the mature stages of differentiation (Dorken, B. et al., J. Immunol.136:4470-4479 (1986)). The predominant form of CD22 in humans isCD22beta which contains seven immunoglobulin superfamily domains in theextracellular domain (FIG. 1) (Wilson, G. L. et al., J. Exp. Med.173:137-146 (1991)). A variant form, CD22 alpha, lacks immunoglobulinsuperfamily domains 3 and 4 (Stamenkovic, I. and Seed, B., Nature345:74-77 (1990)). Ligand-binding to human CD22 has been shown to beassociated with immunoglobulin superfamily domains 1 and 2 (alsoreferred to as epitopes 1 and 2) (Engel, P. et al., J. Exp. Med.181:1581-1586, 1995).

In B-cell NHL, CD22 expression ranges from 91% to 99% in the aggressiveand indolent populations, respectively (Cesano, A. et al., Blood100:350a (2002)). CD22 may function both as a component of the B-cellactivation complex (Sato, S. et al., Semin. Immunol. 10:287-296 (1998))and as an adhesion molecule (Engel, Pl t al., J. Immunol. 150:4719-4732(1993)). The B cells of CD22-deficient mice have a shorter life span andenhanced apoptosis, which suggests a key role of this antigen in B-cellsurvival (Otipoby, K. L. et al., Nature (Lond) 384:634-637 (1996)).After binding with its natural ligand(s) or antibodies, CD22 is rapidlyinternalized, providing a potent costimulatory signal in primary B cellsand proapoptotic signals in neoplastic B cells (Sato, S. et al.,Immunity 5:551-562 (1996)).

Anti-CD22 antibodies have been studied as potential therapies for B cellcancers and other B cell proliferative diseases. Such anti-CD22antibodies include RFB4 Mansfield, E. et al., Blood 90:2020-2026(1997)), CMC-544 (DiJoseph, J. F., Blood 103:1807-1814 (2004)) and LL2(Pawlak-Byczkowska, E. J. et al., Cancer Res. 49:4568-4577 (1989)). TheLL2 antibody (formerly called HPB-2) is an IgG2a mouse monoclonalantibody directed against the CD22 antigen (Pawlak-Byczkowska, E. J. etal. (1989), supra). In vitro immunohistological evaluations demonstratedreactivity of the LL2 antibody with 50 of 51 B-cell NHL specimenstested, but not with other malignancies or normal nonlymphoid tissues(Pawlak-Byczkowska (1989), supra; Stein, R. et al., Cancer Immunol.Immunother. 37:293-298 (1993)).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., CancerImmunol. Immunother. 21:183-87 (1986)). Toxins used in antibody-toxinconjugates include bacterial toxins such as diphtheria toxin, planttoxins such as ricin, small molecule toxins such as geldanamycin (Kerret al (1997) Bioconjugate Chem. 8(6):781-784; Mandler et al (2000)Journal of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000)Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002)Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al.,(1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lodeet al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.53:3336-3342). The toxins may effect their cytotoxic and cytostaticeffects by mechanisms including tubulin binding, DNA binding, ortopoisomerase inhibition (Meyer, D. L. and Senter, P. D. “RecentAdvances in Antibody Drug Conjugates for Cancer Therapy” in AnnualReports in Medicinal Chemistry, Vol 38 (2003) Chapter 23, 229-237). Somecytotoxic drugs tend to be inactive or less active when conjugated tolarge antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is being developed for the treatment of cancers thatexpress CanAg antigen, such as colon, pancreatic, gastric, and others.MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibodydrug conjugate composed of the anti-prostate specific membrane antigen(PSMA) monoclonal antibody linked to the maytansinoid drug moiety, DM1,is under development for the potential treatment of prostate tumors. Thesame maytansinoid drug moiety, DM1, was linked through a non-disulfidelinker, SMCC, to a mouse murine monoclonal antibody, TA.1 (Chari et al.(1992) Cancer Research 52:127-131). This conjugate was reported to be200-fold less potent than the corresponding disulfide linker conjugate.The SMCC linker was considered therein to be “noncleavable.”

Several short peptidic compounds have been isolated from the marinemollusk, Dolabella auricularia, and found to have biological activity(Pettit et al (1993) Tetrahedron 49:9151; Nakamura et al (1995)Tetrahedron Letters 36:5059-5062; Sone et al (1995) Journal Org. Chem.60:4474). Analogs of these compounds have also been prepared, and somewere found to have biological activity (for a review, see Pettit et al(1998) Anti-Cancer Drug Design 13:243-277). For example, auristatin E(U.S. Pat. No. 5,635,483) is a synthetic analogue of the marine naturalproduct Dolastatin 10, an agent that inhibits tubulin polymerization bybinding to the same site on tubulin as the anticancer drug vincristine(G. R. Pettit, (1997) Prog. Chem. Org. Nat. Prod. 70:1-79). Dolastatin10, auristatin PE, and auristatin E are linear peptides having fouramino acids, three of which are unique to the dolastatin class ofcompounds, and a C-terminal amide.

The auristatin peptides, auristain E (AE) and monomethylauristatin(MMAE), synthetic analogs of dolastatin, were conjugated to: (i)chimeric monoclonal antibodies cBR96 (specific to Lewis Y oncarcinomas); (ii) cAC10 which is specific to CD30 on hematologicalmalignancies (Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773; Doronina et al (2003) Nature Biotechnology 21(7):778-784;“Monomethylvaline Compounds Capable of Conjugation to Ligands”;Francisco et al (2003) Blood 102(4):1458-1465; US 2004/0018194; (iii)anti-CD20 antibodies such as Rituxan® (rituximab) (WO 04/032828) for thetreatment of CD20-expressing cancers and immune disorders; (iv)anti-EphB2 antibodies 2H9 and anti-IL-8 for treatment of colorectalcancer (Mao, et al (2004) Cancer Research 64(3):781-788); (v) E-selectinantibody (Bhaskar et al (2003) Cancer Res. 63:6387-6394); and (vi) otheranti-CD30 antibodies (WO 03/043583). Monomethylauristatin (MMAE) hasalso been conjugated to 2H9, an antibody against EphB2R which is a type1 TM tyrosine kinase receptor with close homology between mouse andhuman, and is over-expressed in colorectal cancer cells (Mao et al(2004) Cancer Res. 64:781-788).

Monomethylauristatin MMAF, a variant of auristatin E (MMAE) with aphenylalanine at the C-terminus (U.S. Pat. No. 5,767,237; U.S. Pat. No.6,124,431), has been reported to be less potent than MMAE, but morepotent when conjugated to monoclonal antibodies (Senter et al,Proceedings of the American Association for Cancer Research, Volume 45,Abstract Number 623, presented Mar. 28, 2004). Auristatin F phenylenediamine (AFP); a phenylalanine variant of MMAE was linked to ananti-CD70 mAb, 1F6, through the C-terminus of 1F6 via a phenylenediamine spacer (Law et al, Proceedings of the American Association forCancer Research, Volume 45, Abstract Number 625, presented Mar. 28,2004).

Anti-CD22 antibody-toxin conjugates have also been studied as potentialtherapeutic compounds. For example, early reports described ricin Achain-containing immunotoxins directed against anti-CD22 as potentialanti-cancer agents (May, R. D. et al., Chemical Abstracts106(21):168656xpages 35-36 (1987); Ghetie, M. A. et al., Cancer Research48:2610-2617 (1988); and Amlot, P. L. et al., Blood 82(9):2624-2633(1993)). Where the toxin was a radioisotope, Epratuzumab, the humanized(CDR-grafted) IgG1 version of LL2, has shown evidence of therapeuticactivity for the radioimmunoconjugate (Juweid, M. E. et al., Clin.Cancer Res. 5 (Suppl 10):3292s-3303s (1999); Griffiths, G. L. et al., J.Nucl. Med. 44:77-84 (2003); Linden, O. et al., Clin. Cancer Res. 5(suppl10):3287s-3291s (1999)).

There exists a need in the art for additional drugs to treat various Bcell-related cancers such as lymphomas such as non-Hodgkin's lymphomaand other B cell proliferative disorders. Particularly useful drugs forthis purpose include B cell targeted anti-CD22 antibody-drug conjugateshaving a significantly lower toxicity, yet useful therapeuticefficiency. These and other limitations and problems of the past areaddressed by the present invention.

The recitation of any reference in this application is not an admissionthat the reference is prior art to this application. All referencescited herein, including patents, patent applications and publications,are incorporated by reference in their entirety.

SUMMARY

The invention provides anti-CD22 antibodies and methods of using thesame.

In one aspect, an antibody that binds to CD22 is provided, wherein theantibody comprises at least one, two, three, four, five, or six HVRsselected from:

(1) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;

(2) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;

(3) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:6;

(4) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:10;

(5) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:12; and

(6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:14.

In another aspect, an antibody that binds to CD22 comprises (a) anHVR-L1 comprising the amino acid sequence of SEQ ID NO:10, and (b) atleast one, two, three, four or five HVRs selected from:

(1) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;

(2) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;

(3) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:6;

(4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:12; and

(6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:14.

In another aspect, an antibody that binds to CD22 comprises (a) anHVR-L1 comprising the amino acid sequence of SEQ ID NO:9, and (b) atleast one, two, three, four or five HVRs selected from:

(1) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;

(2) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;

(3) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:6;

(4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:12; and

(6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:14.

In another aspect, an antibody that binds to CD22 comprises (a) anHVR-H3 comprising the amino acid sequence of SEQ ID NO:6, and (b) atleast one, two, three, four, or five HVRs selected from:

(1) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;

(2) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;

(3) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:9;

(4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:12; and

(5) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:14.

In another aspect, an antibody that binds to CD22 comprises (a) anHVR-H3 comprising the amino acid sequence of SEQ ID NO:6, and (b) atleast one, two, three, four, or five HVRs selected from:

(1) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;

(2) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;

(3) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:10;

(4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:12; and

(5) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:14.

In one embodiment, the antibody comprises an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:10. In one embodiment, the antibody furthercomprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4. In oneembodiment, the antibody further comprises an HVR-L2 comprising theamino acid sequence of SEQ NO:12 and an HVR-L3 comprising the amino acidsequence of SEQ ID NO:14.

In certain embodiments, any of the above antibodies further comprises atleast one framework selected from a VH subgroup III consensus frameworkand a VL subgroup I consensus framework.

In one aspect, an antibody that binds to CD22 is provided, wherein theantibody comprises a heavy chain variable domain having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto an amino acid sequence of SEQ ID NO:16. In one embodiment, theantibody comprises a heavy chain variable domain of SEQ ID NO:16.

In one aspect, the antibody further comprises a light chain variabledomain having at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to an amino acid sequence of SEQ ID NO:17.In one embodiment, the antibody comprises a light chain variable domainof SEQ ID NO:17.

In one aspect, the antibody further comprises a light chain variabledomain having at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to an amino acid sequence of SEQ ID NO:18.In one embodiment, the antibody comprises a light chain variable domainof SEQ ID NO:18.

In one embodiment, the antibody comprises a heavy chain variable domainhaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO:16and a light chain variable domain having at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to anamino acid sequence of SEQ ID NO:17. In one embodiment, the antibodycomprises a heavy chain variable domain having at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto an amino acid sequence of SEQ ID NO:16 and a light chain variabledomain having at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to an amino acid sequence of SEQ IDNO:18. In one embodiment, the heavy chain variable domain comprises theamino acid sequence of SEQ ID NO:16, and the light chain variable domaincomprises the amino acid sequence of SEQ ID NO:17. In one embodiment,the heavy chain variable domain comprises the amino acid sequence of SEQID NO:16, and the light chain variable domain comprises the amino acidsequence of SEQ ID NO:18.

In certain embodiments, a polynucleotide encoding any of the aboveantibodies is provided. In one embodiment, a vector comprising thepolynucleotide is provided. In one embodiment, a host cell comprisingthe vector is provided. In one embodiment, the host cell is eukaryotic.In one embodiment, the host cell is a Chinese hamster ovary (CHO) cell.

In one embodiment, a method of making an anti-CD22 antibody is provided,wherein the method comprises culturing the host cell under conditionssuitable for expression of the polynucleotide encoding the antibody, andisolating the antibody.

In one aspect, an antibody that binds to CD22 expressed on the surfaceof a cell is provided. In one embodiment, the antibody binds to anepitope within a region of human or mouse CD22 comprising domain 1 ordomain 2 or domains 1 and 2. In one embodiment, the cell is mammaliancell. In one embodiment, the cell is a human cell. In one embodiment,the cell is a cancer cell. In one embodiment the cell is a B cell. Inone embodiment the cancer cell is a B cell.

In certain embodiments, any of the above antibodies is a monoclonalantibody. In one embodiment, the antibody is an antibody fragmentselected from a Fab, Fab′-SH, Fv, scFv, or (Fab′)₂ fragment. In oneembodiment, the antibody is humanized. In one embodiment, the antibodyis human.

In one aspect, a method of detecting the presence of CD22 in abiological sample is provided, the method comprising contacting thebiological sample with any of the above antibodies under conditionspermissive for binding of the antibody to CD22, and detecting whether acomplex is formed between the antibody and CD22. In one embodiment, thebiological sample comprises B cells. In one embodiment, the biologicalsample is from a mammal experiencing or suspected of experiencing a Bcell disorder and/or a B cell proliferative disorder including, but notlimited to, lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL,relapsed aggressive NHL, relapsed indolent NHL, refractory NHL,refractory indolent NHL, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acutelymphocytic leukemia (ALL), and mantle cell lymphoma.

In one aspect, a method of diagnosing a cell proliferative disorderassociated with increased expression of CD22 is provided, the methodcomprising contacting a test cell with any of the above antibodies;determining the level of expression of CD22 by detecting binding of theantibody to CD22; and comparing the level of expression of CD22 by thetest cell with the level of expression of CD22 by a control cell,wherein a higher level of expression of CD22 by the test cell ascompared to the control cell indicates the presence of a cellproliferative disorder associated with increased expression of CD22. Inone embodiment, the test cell is a cell from a patient suspected ofhaving a cell proliferative disorder, such as a B-cell proliferativedisorder. In one embodiment, the cell proliferative disorder is selectedfrom B cell disorders including but not limited to lymphoma,non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL,relapsed indolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma. In one embodiment, the method comprises determining the levelof expression of CD22 on the surface of the test cell and comparing thelevel of expression of CD22 on the surface of the test cell with thelevel of expression of CD22 on the surface of the control cell.

In one aspect, a method of diagnosing a cell proliferative disorderassociated with an increase in cells, such as B cells, expressing CD22is provided, the method comprising contacting a test cells in abiological sample with any of the above antibodies; determining thelevel of antibody bound to test cells in the sample by detecting bindingof the antibody to CD22; and comparing the level of antibody bound tocells in a control sample, wherein the level of antibody bound isnormalized to the number of CD22-expressing cells in the test andcontrol samples, and wherein a higher level of antibody bound in thetest sample as compared to the control sample indicates the presence ofa cell proliferative disorder associated with cells expressing CD22.

In one aspect, a method of detecting soluble CD22 in blood or serum, themethod comprising contacting a test sample of blood or serum from amammal suspected of experiencing a B cell proliferative disorder with ananti-CD22 antibody of the invention and detecting a increase in solubleCD22 in the test sample relative to a control sample of blood or serumfrom a normal mammal. In an embodiment, the method of detecting isuseful as a method of diagnosing a B cell proliferative disorderassociated with an increase in soluble CD22 in blood or serum of amammal.

In one aspect, the antibodies of the invention include cysteineengineered antibodies where one or more amino acids of a parent antibodyare replaced with a free cysteine amino acid as disclosed inWO2006/034488 (herein incorporated by reference in its entirety). Anyform of anti-CD22 antibody may be so engineered, i.e. mutated. Forexample, a parent Fab antibody fragment may be engineered to form acysteine engineered Fab, referred to herein as “ThioFab.” Similarly, aparent monoclonal antibody may be engineered to form a “ThioMab.” Itshould be noted that a single site mutation yields a single engineeredcysteine residue in a ThioFab, while a single site mutation yields twoengineered cysteine residues in a ThioMab, due to the dimeric nature ofthe IgG antibody. The cysteine engineered anti-CD22 antibodies of theinvention include monoclonal antibodies, humanized or chimericmonoclonal antibodies, and antigen-binding fragments of antibodies,fusion polypeptides and analogs that preferentially bind cell-associatedCD22 polypeptides. A cysteine engineered antibody may alternativelycomprise an antibody comprising a cysteine at a position disclosedherein in the antibody or Fab, resulting from the sequence design and/orselection of the antibody, without necessarily altering a parentantibody, such as by phage display antibody design and selection orthrough de novo design of light chain and/or heavy chain frameworksequences and constant regions. A cysteine engineered antibody comprisesone or more free cysteine amino acids having a thiol reactivity value inthe ranges of 0.6 to 1.0; 0.7 to 1.0 or 0.8 to 1.0. A free cysteineamino acid is a cysteine residue which has been engineered into theparent antibody and is not part of a disulfide bridge. Cysteineengineered antibodies are useful for attachment of cytotoxic and/orimaging compounds at the site of the engineered cysteine through, forexample, a maleimide or haloacetyl. The nucleophilic reactivity of thethiol functionality of a Cys residue to a maleimide group is about 1000times higher compared to any other amino acid functionality in aprotein, such as amino group of lysine residues or the N-terminal aminogroup. Thiol specific functionality in iodoacetyl and maleimide reagentsmay react with amine groups, but higher pH (>9.0) and longer reactiontimes are required (Garman, 1997, Non-Radioactive Labelling: A PracticalApproach, Academic Press, London).

In an embodiment, a cysteine engineered anti-CD22 antibody of theinvention comprises an engineered cysteine at any one of the followingpositions, where the position is number according to Kabat et al. in thelight chain (see Kabat et al (1991) Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md.) and according to EU numbering inthe heavy chain (including the Fc region) (see see Kabat et al. (1991),supra), wherein the light chain constant region depicted by underliningin FIG. 17A begins at position 108 (Kabat numbering) and the heavy chainconstant region depicted by underling in FIGS. 17B and 17C begins atposition 118 (EU numbering). The position may also be referred to by itsposition in sequential numbering of the amino acids of the full lengthlight chain or heavy chain shown in FIGS. 17A-17C. According to oneembodiment of the invention, an anti-CD22 antibody comprises anengineered cysteine at LC-V205C (Kabat number: Val 205; sequentialnumber 210 in FIG. 17A engineered to be Cys at that position). Theengineered cysteine in the light chain is shown in bold, doubleunderlined text in FIG. 17A. According to one embodiment, an anti-CD22antibody comprises an engineered cysteine at HC-A118C (EU number: Ala118; sequential number 121 in FIG. 17B engineered to be Cys at thatposition). The engineered cysteine in the heavy chain is shown in bold,double underlined text in FIG. 17B. According to one embodiment, ananti-CD22 antibody comprises an engineered cysteine at Fc-S400C (EUnumber: Ser 400; sequential number 403 in FIG. 17C engineered to be Cysat that position). The engineered cysteine in the Fc region of the heavychain is shown in bold, double underlined text in FIG. 17C. In otherembodiments, the engineered cysteine of the heavy chain (including theFc region) is at any one of the following positions (according to EUnumbering): 41, 88, 116, 118, 120, 171, 282, 375, or 400. Thus, changesin the amino acid at these positions for a parent anti-CD22 antibody ofthe invention are: A41C, A88C, S116C, A118C, T120C, A171C, V282C, S375C,or S400C. In other embodiments, the engineered cysteine of the lightchain is at any one of the following positions (according to Kabatnumbering): 15, 43, 110, 144, 168, 205. Thus, changes in the amino acidat these positions for a parent anti-CD22 antibody of the invention are:V15C, A43C, V110C, A144C, S168C, or V205C.

A cysteine engineered anti-CD22 antibody comprises one or more freecysteine amino acids wherein the cysteine engineered anti-CD22 antibodybinds to a CD22 polypeptide and is prepared by a process comprisingreplacing one or more amino acid residues of a parent anti-CD22 antibodyby cysteine wherein the parent antibody comprises at least one HVRsequence selected from:

(a) an HVR-L1 sequence RSSQSIVHSNGNTFLE (SEQ ID NO:9) or sequenceRSSQSIVHSVGNTFLE (SEQ ID NO:10) (FIG. 2B);

(b) an HVR-L2 sequence KVSNRFS SEQ ID NO:12 (FIG. 2B);

(c) an HVR-L3 sequence FQGSQFPYT (SEQ ID NO:14) (FIG. 2B);

(d) an HVR-H1 sequence GYEFSRSWMN (SEQ ID NO:2) (FIG. 2A);

(e) an HVR-H2 sequence GRIYPGDGDTNYSGKFKG (SEQ ID NO:4 (FIG. 2A); and

(f) an HVR-H3 sequence DGSSWDWYFDV (SEQ ID NO:6) (FIG. 2A).

In a certain aspect, the invention concerns a cysteine engineeredanti-CD22 antibody, comprising an amino acid sequence having at leastabout 80% amino acid sequence identity, alternatively at least about81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, to acysteine engineered antibody having a full-length amino acid sequence asdisclosed herein, or a cysteine engineered antibody amino acid sequencelacking the signal peptide as disclosed herein.

In a yet further aspect, the invention concerns an isolated cysteineengineered anti-CD22 antibody comprising an amino acid sequence that isencoded by a nucleotide sequence that hybridizes to the complement of aDNA molecule encoding (a) a cysteine engineered antibody having afull-length amino acid sequence as disclosed herein, (b) a cysteineengineered antibody amino acid sequence lacking the signal peptide asdisclosed herein, (c) an extracellular domain of a transmembranecysteine engineered antibody protein, with or without the signalpeptide, as disclosed herein, (d) an amino acid sequence encoded by anyof the nucleic acid sequences disclosed herein or (e) any otherspecifically defined fragment of a full-length cysteine engineeredantibody amino acid sequence as disclosed herein.

In a specific aspect, the invention provides an isolated cysteineengineered anti-CD22 antibody without the N-terminal signal sequenceand/or without the initiating methionine and is encoded by a nucleotidesequence that encodes such an amino acid sequence as described in.Processes for producing the same are also herein described, whereinthose processes comprise culturing a host cell comprising a vector whichcomprises the appropriate encoding nucleic acid molecule underconditions suitable for expression of the cysteine engineered antibodyand recovering the cysteine engineered antibody from the cell culture.

Another aspect of the invention provides an isolated cysteine engineeredanti-CD22 antibody which is either transmembrane domain-deleted ortransmembrane domain-inactivated. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thecysteine engineered antibody and recovering the cysteine engineeredantibody from the cell culture.

In other embodiments, the invention provides isolated anti-CD22 chimericcysteine engineered antibodies comprising any of the herein describedcysteine engineered antibody fused to a heterologous (non-CD22)polypeptide. Example of such chimeric molecules comprise any of theherein described cysteine engineered antibodies fused to a heterologouspolypeptide such as, for example, an epitope tag sequence or a Fc regionof an immunoglobulin.

The cysteine engineered anti-CD22 antibody may be a monoclonal antibody,antibody fragment, chimeric antibody, humanized antibody, single-chainantibody or antibody that competitively inhibits the binding of ananti-CD22 polypeptide antibody to its respective antigenic epitope.Antibodies of the present invention may optionally be conjugated to agrowth inhibitory agent or cytotoxic agent such as a toxin, including,for example, an auristatin, an antibiotic, a radioactive isotope, anucleolytic enzyme, or the like. The antibodies of the present inventionmay optionally be produced in CHO cells or bacterial cells andpreferably inhibit the growth or proliferation of or induce the death ofa cell to which they bind. For diagnostic purposes, the antibodies ofthe present invention may be detectably labeled, attached to a solidsupport, or the like.

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein described anti-CD22antibodies and anti-CD22 cysteine engineered antibodies. Host cellscomprising any such vector are also provided. By way of example, thehost cells may be CHO cells, E. coli cells, or yeast cells. A processfor producing any of the herein described polypeptides is furtherprovided and comprises culturing host cells under conditions suitablefor expression of the desired polypeptide and recovering the desiredpolypeptide from the cell culture.

Cysteine engineered antibodies may be useful in the treatment of cancerand include antibodies specific for cell surface and transmembranereceptors, and tumor-associated antigens (TAA). Such antibodies may beused as naked antibodies (unconjugated to a drug or label moiety) or asantibody-drug conjugates (ADC). Cysteine engineered antibodies of theinvention may be site-specifically and efficiently coupled with athiol-reactive reagent. The thiol-reactive reagent may be amultifunctional linker reagent, a capture label reagent, a fluorophorereagent, or a drug-linker intermediate. The cysteine engineered antibodymay be labeled with a detectable label, immobilized on a solid phasesupport and/or conjugated with a drug moiety. Thiol reactivity may begeneralized to any antibody where substitution of amino acids withreactive cysteine amino acids may be made within the ranges in the lightchain selected from amino acid ranges: L-10 to L-20; L-38 to L-48; L-105to L-115; L-139 to L-149; L-163 to L-173; and within the ranges in theheavy chain selected from amino acid ranges: H-35 to H-45; H-83 to H-93;H-114 to H-127; and H-170 to H-184, and in the Fc region within theranges selected from H-268 to H-291; H-319 to H-344; H-370 to H-380; andH-395 to H-405, where the numbering of amino acid positions begins atposition 1 of the Kabat numbering system (Kabat et al. (1991) Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.) and continues sequentiallythereafter as disclosed in WO2006034488. Thiol reactivity may also begeneralized to certain domains of an antibody, such as the light chainconstant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3.Cysteine replacements resulting in thiol reactivity values of 0.6 andhigher may be made in the heavy chain constant domains α, δ, ε, γ, and μof intact antibodies: IgA, IgD, IgE, IgG, and IgM, respectively,including the IgG subclasses: IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.Such antibodies and their uses are disclosed in WO2006/034488.

Cysteine engineered antibodies of the invention preferably retain theantigen binding capability of their wild type, parent antibodycounterparts. Thus, cysteine engineered antibodies are capable ofbinding, preferably specifically, to antigens. Such antigens include,for example, tumor-associated antigens (TAA), cell surface receptorproteins and other cell surface molecules, transmembrane proteins,signalling proteins, cell survival regulatory factors, cellproliferation regulatory factors, molecules associated with (for e.g.,known or suspected to contribute functionally to) tissue development ordifferentiation, lymphokines, cytokines, molecules involved in cellcycle regulation, molecules involved in vasculogenesis and moleculesassociated with (for e.g., known or suspected to contribute functionallyto) angiogenesis. The tumor-associated antigen may be a clusterdifferentiation factor (i.e., a CD protein, including but not limited toCD22). Cysteine engineered anti-CD22 antibodies of the invention retainthe antigen binding capability of their parent anti-CD22 antibodycounterparts. Thus, cysteine engineered anti-CD22 antibodies of theinvention are capable of binding, preferably specifically, to CD22antigens including human anti-CD22 isoforms beta and/or alpha, includingwhen such antigens are expressed on the surface of cells, including,without limitation, B cells.

An antibody of the invention may be conjugated to other thiol-reactiveagents in which the reactive group is, for example, a maleimide, aniodoacetamide, a pyridyl disulfide, or other thiol-reactive conjugationpartner (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probesand Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992,Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: APractical Approach, Academic Press, London; Means (1990) BioconjugateChem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) AcademicPress, San Diego, pp. 40-55, 643-671). The partner may be a cytotoxicagent (e.g. a toxin such as doxorubicin or pertussis toxin), afluorophore such as a fluorescent dye like fluorescein or rhodamine, achelating agent for an imaging or radiotherapeutic metal, a peptidyl ornon-peptidyl label or detection tag, or a clearance-modifying agent suchas various isomers of polyethylene glycol, a peptide that binds to athird component, or another carbohydrate or lipophilic agent.

In one aspect, antibodies of the invention may be conjugated with anylabel moiety which can be covalently attached to the antibody through areactive moiety, an activated moiety, or a reactive cysteine thiol group(Singh et al (2002) Anal. Biochem. 304:147-15; Harlow E. and Lane, D.(1999) Using Antibodies: A Laboratory Manual, Cold Springs HarborLaboratory Press, Cold Spring Harbor, N.Y.; Lundblad R. L. (1991)Chemical Reagents for Protein Modification, 2nd ed. CRC Press, BocaRaton, Fla.). The attached label may function to: (i) provide adetectable signal; (ii) interact with a second label to modify thedetectable signal provided by the first or second label, e.g. to giveFRET (fluorescence resonance energy transfer); (iii) stabilizeinteractions or increase affinity of binding, with antigen or ligand;(iv) affect mobility, e.g. electrophoretic mobility orcell-permeability, by charge, hydrophobicity, shape, or other physicalparameters, or (v) provide a capture moiety, to modulate ligandaffinity, antibody/antigen binding, or ionic complexation.

Labelled cysteine engineered antibodies may be useful in diagnosticassays, e.g., for detecting expression of an antigen of interest inspecific cells, tissues, or serum. For diagnostic applications, theantibody will typically be labeled with a detectable moiety. Numerouslabels are available which can be generally grouped into the followingcategories:

Radioisotopes (radionuclides), such as ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At,or ²¹³Bi. Radioisotope labelled antibodies are useful in receptortargeted imaging experiments. The antibody can be labeled with ligandreagents that bind, chelate or otherwise complex a radioisotope metalwhere the reagent is reactive with the engineered cysteine thiol of theantibody, using the techniques described in Current Protocols inImmunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, NewYork, N.Y., Pubs. (1991). Chelating ligands which may complex a metalion include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas,Tex.). Radionuclides can be targeted via complexation with theantibody-drug conjugates of the invention (Wu et al (2005) NatureBiotechnology 23(9):1137-1146).

Linker reagents such as DOTA-maleimide(4-maleimidobutyramidobenzyl-DOTA) can be prepared by the reaction ofaminobenzyl-DOTA with 4-maleimidobutyric acid (Fluka) activated withisopropylchloroformate (Aldrich), following the procedure of Axworthy etal (2000) Proc. Natl. Acad. Sci. USA 97(4):1802-1807). DOTA-maleimidereagents react with the free cysteine amino acids of the cysteineengineered antibodies and provide a metal complexing ligand on theantibody (Lewis et al (1998) Bioconj. Chem. 9:72-86). Chelating linkerlabelling reagents such as DOTA-NHS(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) are commercially available (Macrocyclics,Dallas, Tex.). Receptor target imaging with radionuclide labelledantibodies can provide a marker of pathway activation by detection andquantitation of progressive accumulation of antibodies in tumor tissue(Albert et al (1998) Bioorg. Med. Chem. Lett. 8:1207-1210). Theconjugated radio-metals may remain intracellular following lysosomaldegradation.

Metal-chelate complexes suitable as antibody labels for imagingexperiments are disclosed: U.S. Pat. No. 5,342,606; U.S. Pat. No.5,428,155; U.S. Pat. No. 5,316,757; U.S. Pat. No. 5,480,990; U.S. Pat.No. 5,462,725; U.S. Pat. No. 5,428,139; U.S. Pat. No. 5,385,893; U.S.Pat. No. 5,739,294; U.S. Pat. No. 5,750,660; U.S. Pat. No. 5,834,456;Hnatowich et al (1983) J. Immunol. Methods 65:147-157; Meares et al(1984) Anal. Biochem. 142:68-78; Mirzadeh et al (1990) BioconjugateChem. 1:59-65; Meares et al (1990) J. Cancer 1990, Suppl. 10:21-26;Izard et al (1992) Bioconjugate Chem. 3:346-350; Nikula et al (1995)Nucl. Med. Biol. 22:387-90; Camera et al (1993) Nucl. Med. Biol.20:955-62; Kukis et al (1998) J. Nucl. Med. 39:2105-2110; Verel et al(2003) J. Nucl. Med. 44:1663-1670; Camera et al (1994) J. Nucl. Med.21:640-646; Ruegg et al (1990) Cancer Res. 50:4221-4226; Verel et al(2003) J. Nucl. Med. 44:1663-1670; Lee et al (2001) Cancer Res.61:4474-4482; Mitchell, et al (2003) J. Nucl. Med. 44:1105-1112;Kobayashi et al (1999) Bioconjugate Chem. 10:103-111; Miederer et al(2004) J. Nucl. Med. 45:129-137; DeNardo et al (1998) Clinical CancerResearch 4:2483-90; Blend et al (2003) Cancer Biotherapy &Radiopharmaceuticals 18:355-363; Nikula et al (1999) J. Nucl. Med.40:166-76; Kobayashi et al (1998) J. Nucl. Med. 39:829-36; Mardirossianet al (1993) Nucl. Med. Biol. 20:65-74; Roselli et al (1999) CancerBiotherapy & Radiopharmaceuticals, 14:209-20.

(b) Fluorescent labels such as rare earth chelates (europium chelates),fluorescein types including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine types including TAMRA; dansyl; Lissamine;cyanines; phycoerythrins; Texas Red; and analogs thereof. Thefluorescent labels can be conjugated to antibodies using the techniquesdisclosed in Current Protocols in Immunology, supra, for example.Fluorescent dyes and fluorescent label reagents include those which arecommercially available from Invitrogen/Molecular Probes (Eugene, Oreg.)and Pierce Biotechnology, Inc. (Rockford, Ill.).

(c) Various enzyme-substrate labels are available or disclosed (U.S.Pat. No. 4,275,149). The enzyme generally catalyzes a chemicalalteration of a chromogenic substrate that can be measured using varioustechniques. For example, the enzyme may catalyze a color change in asubstrate, which can be measured spectrophotometrically. Alternatively,the enzyme may alter the fluorescence or chemiluminescence of thesubstrate. Techniques for quantifying a change in fluorescence aredescribed above. The chemiluminescent substrate becomes electronicallyexcited by a chemical reaction and may then emit light which can bemeasured (using a chemiluminometer, for example) or donates energy to afluorescent acceptor. Examples of enzymatic labels include luciferases(e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No.4,737,456), luciferin, 2,3-dihydrophthalazinediones, malatedehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP),alkaline phosphatase (AP), β-galactosidase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Techniques for conjugating enzymes to antibodies are described inO'Sullivan et al (1981) “Methods for the Preparation of Enzyme-AntibodyConjugates for use in Enzyme Immunoassay”, in Methods in Enzym. (ed J.Langone & H. Van Vunakis), Academic Press, New York, 73:147-166.

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRP) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethylbenzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review, see U.S. Pat. No. 4,275,149and U.S. Pat. No. 4,318,980.

A label may be indirectly conjugated with an amino acid side chain, anactivated amino acid side chain, a cysteine engineered antibody, and thelike. For example, the antibody can be conjugated with biotin and any ofthe three broad categories of labels mentioned above can be conjugatedwith avidin or streptavidin, or vice versa. Biotin binds selectively tostreptavidin and thus, the label can be conjugated with the antibody inthis indirect manner. Alternatively, to achieve indirect conjugation ofthe label with the polypeptide variant, the polypeptide variant isconjugated with a small hapten (e.g., digoxin) and one of the differenttypes of labels mentioned above is conjugated with an anti-haptenpolypeptide variant (e.g., anti-digoxin antibody). Thus, indirectconjugation of the label with the polypeptide variant can be achieved(Hermanson, G. (1996) in Bioconjugate Techniques Academic Press, SanDiego).

The antibody of the present invention may be employed in any known assaymethod, such as ELISA, competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays (Zola, (1987) MonoclonalAntibodies: A Manual of Techniques, pp. 147-158, CRC Press, Inc.).

A detection label may be useful for localizing, visualizing, andquantitating a binding or recognition event. The labelled antibodies ofthe invention can detect cell-surface receptors. Another use fordetectably labelled antibodies is a method of bead-based immunocapturecomprising conjugating a bead with a fluorescent labelled antibody anddetecting a fluorescence signal upon binding of a ligand. Similarbinding detection methodologies utilize the surface plasmon resonance(SPR) effect to measure and detect antibody-antigen interactions.

Detection labels such as fluorescent dyes and chemiluminescent dyes(Briggs et al (1997) “Synthesis of Functionalised Fluorescent Dyes andTheir Coupling to Amines and Amino Acids,” J. Chem. Soc., Perkin-Trans.1:1051-1058) provide a detectable signal and are generally applicablefor labelling antibodies, preferably with the following properties: (i)the labelled antibody should produce a very high signal with lowbackground so that small quantities of antibodies can be sensitivelydetected in both cell-free and cell-based assays; and (ii) the labelledantibody should be photostable so that the fluorescent signal may beobserved, monitored and recorded without significant photo bleaching.For applications involving cell surface binding of labelled antibody tomembranes or cell surfaces, especially live cells, the labels preferably(iii) have good water-solubility to achieve effective conjugateconcentration and detection sensitivity and (iv) are non-toxic to livingcells so as not to disrupt the normal metabolic processes of the cellsor cause premature cell death.

Direct quantification of cellular fluorescence intensity and enumerationof fluorescently labelled events, e.g. cell surface binding ofpeptide-dye conjugates may be conducted on an system (FMAT® 8100 HTSSystem, Applied Biosystems, Foster City, Calif.) that automatesmix-and-read, non-radioactive assays with live cells or beads (Miraglia,“Homogeneous cell- and bead-based assays for high throughput screeningusing fluorometric microvolume assay technology”, (1999) J. ofBiomolecular Screening 4:193-204). Uses of labelled antibodies alsoinclude cell surface receptor binding assays, immunocapture assays,fluorescence linked immunosorbent assays (FLISA), caspase-cleavage(Zheng, “Caspase-3 controls both cytoplasmic and nuclear eventsassociated with Fas-mediated apoptosis in vivo”, (1998) Proc. Natl.Acad. Sci. USA 95:618-23; U.S. Pat. No. 6,372,907), apoptosis (Vermes,“A novel assay for apoptosis. Flow cytometric detection ofphosphatidylserine expression on early apoptotic cells using fluoresceinlabelled Annexin V” (1995) J. Immunol. Methods 184:39-51) andcytotoxicity assays. Fluorometric microvolume assay technology can beused to identify the up or down regulation by a molecule that istargeted to the cell surface (Swartzman, “A homogeneous and multiplexedimmunoassay for high-throughput screening using fluorometric microvolumeassay technology”, (1999) Anal. Biochem. 271:143-51).

Labelled antibodies of the invention are useful as imaging biomarkersand probes by the various methods and techniques of biomedical andmolecular imaging such as: (i) MRI (magnetic resonance imaging); (ii)MicroCT (computerized tomography); (iii) SPECT (single photon emissioncomputed tomography); (iv) PET (positron emission tomography) Chen et al(2004) Bioconjugate Chem. 15:41-49; (v) bioluminescence; (vi)fluorescence; and (vii) ultrasound. Immunoscintigraphy is an imagingprocedure in which antibodies labeled with radioactive substances areadministered to an animal or human patient and a picture is taken ofsites in the body where the antibody localizes (U.S. Pat. No.6,528,624). Imaging biomarkers may be objectively measured and evaluatedas an indicator of normal biological processes, pathogenic processes, orpharmacological responses to a therapeutic intervention. Biomarkers maybe of several types: Type 0 are natural history markers of a disease andcorrelate longitudinally with known clinical indices, e.g. MRIassessment of synovial inflammation in rheumatoid arthritis; Type Imarkers capture the effect of an intervention in accordance with amechanism-of-action, even though the mechanism may not be associatedwith clinical outcome; Type II markers function as surrogate endpointswhere the change in, or signal from, the biomarker predicts a clinicalbenefit to “validate” the targeted response, such as measured boneerosion in rheumatoid arthritis by CT. Imaging biomarkers thus canprovide pharmacodynamic (PD) therapeutic information about: (i)expression of a target protein, (ii) binding of a therapeutic to thetarget protein, i.e. selectivity, and (iii) clearance and half-lifepharmacokinetic data. Advantages of in vivo imaging biomarkers relativeto lab-based biomarkers include: non-invasive treatment, quantifiable,whole body assessment, repetitive dosing and assessment, i.e. multipletime points, and potentially transferable effects from preclinical(small animal) to clinical (human) results. For some applications,bioimaging supplants or minimizes the number of animal experiments inpreclinical studies.

Peptide labelling methods are well known. See Haugland, 2003, MolecularProbes Handbook of Fluorescent Probes and Research Chemicals, MolecularProbes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, (1997)Non-Radioactive Labelling: A Practical Approach, Academic Press, London;Means (1990) Bioconjugate Chem. 1:2; Glazer et al (1975) ChemicalModification of Proteins. Laboratory Techniques in Biochemistry andMolecular Biology (T. S. Work and E. Work, Eds.) American ElsevierPublishing Co., New York; Lundblad, R. L. and Noyes, C. M. (1984)Chemical Reagents for Protein Modification, Vols. I and II, CRC Press,New York; Pfleiderer, G. (1985) “Chemical Modification of Proteins”,Modern Methods in Protein Chemistry, H. Tschesche, Ed., Walter DeGryter,Berlin and New York; and Wong (1991) Chemistry of Protein Conjugationand Cross-linking, CRC Press, Boca Raton, Fla.); De Leon-Rodriguez et al(2004) Chem. Eur. J. 10:1149-1155; Lewis et al (2001) Bioconjugate Chem.12:320-324; Li et al (2002) Bioconjugate Chem. 13:110-115; Mier et al(2005) Bioconjugate Chem. 16:240-237.

Peptides and proteins labelled with two moieties, a fluorescent reporterand quencher in sufficient proximity undergo fluorescence resonanceenergy transfer (FRET). Reporter groups are typically fluorescent dyesthat are excited by light at a certain wavelength and transfer energy toan acceptor, or quencher, group, with the appropriate Stokes shift foremission at maximal brightness. Fluorescent dyes include molecules withextended aromaticity, such as fluorescein and rhodamine, and theirderivatives. The fluorescent reporter may be partially or significantlyquenched by the quencher moiety in an intact peptide. Upon cleavage ofthe peptide by a peptidase or protease, a detectable increase influorescence may be measured (Knight, C. (1995) “Fluorimetric Assays ofProteolytic Enzymes”, Methods in Enzymology, Academic Press, 248:18-34).

The labelled antibodies of the invention may also be used as an affinitypurification agent. In this process, the labelled antibody isimmobilized on a solid phase such a Sephadex resin or filter paper,using methods well known in the art. The immobilized antibody iscontacted with a sample containing the antigen to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the antigento be purified, which is bound to the immobilized polypeptide variant.Finally, the support is washed with another suitable solvent, such asglycine buffer, pH 5.0, that will release the antigen from thepolypeptide variant.

Labelling reagents typically bear reactive functionality which may react(i) directly with a cysteine thiol of a cysteine engineered antibody toform the labelled antibody, (ii) with a linker reagent to form alinker-label intermediate, or (iii) with a linker antibody to form thelabelled antibody. Reactive functionality of labelling reagents include:maleimide, haloacetyl, iodoacetamide succinimidyl ester (e.g. NHS,N-hydroxysuccinimide), isothiocyanate, sulfonyl chloride,2,6-dichlorotriazinyl, pentafluorophenyl ester, and phosphoramidite,although other functional groups can also be used.

An exemplary reactive functional group is N-hydroxysuccinimidyl ester(NHS) of a carboxyl group substituent of a detectable label, e.g. biotinor a fluorescent dye. The NHS ester of the label may be preformed,isolated, purified, and/or characterized, or it may be formed in situand reacted with a nucleophilic group of an antibody. Typically, thecarboxyl form of the label is activated by reacting with somecombination of a carbodiimide reagent, e.g. dicyclohexylcarbodiimide,diisopropylcarbodiimide, or a uronium reagent, e.g. TSTU(O—(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, HBTU(O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate),or HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), an activator, such as 1-hydroxybenzotriazole(HOBt), and N-hydroxysuccinimide to give the NHS ester of the label. Insome cases, the label and the antibody may be coupled by in situactivation of the label and reaction with the antibody to form thelabel-antibody conjugate in one step. Other activating and couplingreagents include TBTU(2-(1H-benzotriazo-1-yl)-1-1,3,3-tetramethyluroniumhexafluorophosphate), TFFH(N,N′,N″,N′″-tetramethyluronium 2 PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate, EEDQ(2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline), DCC(dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT(1-(mesitylene-2 and aryl sulfonyl halides, e.g.triisopropylbenzenesulfonyl chloride.

Albumin Binding Peptide-Fab Compounds of the Invention:

In one aspect, the antibody of the invention is fused to an albuminbinding protein. Plasma-protein binding can be an effective means ofimproving the pharmacokinetic properties of short lived molecules.Albumin is the most abundant protein in plasma. Serum albumin bindingpeptides (ABP) can alter the pharmacodynamics of fused active domainproteins, including alteration of tissue uptake, penetration, anddiffusion. These pharmacodynamic parameters can be modulated by specificselection of the appropriate serum albumin binding peptide sequence (US20040001827). A series of albumin binding peptides were identified byphage display screening (Dennis et al. (2002) “Albumin Binding As AGeneral Strategy For Improving The Pharmacokinetics Of Proteins” J Biol.Chem. 277:35035-35043; WO 01/45746). Compounds of the invention includeABP sequences taught by: (i) Dennis et al (2002) J Biol. Chem.277:35035-35043 at Tables III and IV, page 35038; (ii) US 20040001827 at[0076] SEQ ID NOS: 9-22; and (iii) WO 01/45746 at pages 12-13, all ofwhich are incorporated herein by reference. Albumin Binding (ABP)-Fabsare engineered by fusing an albumin binding peptide to the C-terminus ofFab heavy chain in 1:1 stoichiometric ratio (1 ABP/1 Fab). It was shownthat association of these ABP-Fabs with albumin increased antibody halflife by more than 25 fold in rabbits and mice. The above describedreactive Cys residues can therefore be introduced in these ABP-Fabs andused for site-specific conjugation with cytotoxic drugs followed by invivo animal studies.

Exemplary albumin binding peptide sequences include, but are not limitedto the amino acid sequences listed in SEQ ID NOS:42-46:

SEQ ID NO: 42 CDKTHTGGGSQRLMEDICLPRWGCLWEDDF SEQ ID NO: 43QRLMEDICLPRWGCLWEDDF SEQ ID NO: 44 QRLIEDICLPRWGCLWEDDF SEQ ID NO: 45RLIEDICLPRWGCLWEDD SEQ ID NO: 46 DICLPRWGCLW

Antibody-Drug Conjugates

In another aspect, the invention provides immunoconjugates, orantibody-drug conjugates (ADC), comprising an antibody conjugated to acytotoxic agent such as a chemotherapeutic agent, a drug, a growthinhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate). In another aspect, theinvention further provides methods of using the immunoconjugates. In oneaspect, an immunoconjugate comprises any of the above anti-CD22antibodies covalently attached to a cytotoxic agent or a detectableagent.

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Thetoxins may effect their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784)and are under therapeutic development.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein. Enzymatically active toxins and fragments thereof thatcan be used include diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. See, e.g., WO 93/21232 published Oct.28, 1993. A variety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2 propionate (SPDP), iminothiolane (IT), bifunctionalderivatives of imidoesters (such as dimethyl adipimidate HCl), activeesters (such as disuccinimidyl suberate), aldehydes (such asglutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al (1987) Science, 238:1098.Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody (WO94/11026).

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, auristatins, atrichothecene, and CC 1065, and the derivatives of these toxins thathave toxin activity, are also contemplated herein.

Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) of the invention conjugated to one or moremaytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol andmaytansinol analogues prepared synthetically according to known methods.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides). and those having modifications at otherpositions

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H₂S or P₂S₅);C-14-alkoxymethyl(demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15 (U.S. Pat. No. 4,364,866)(prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Exemplary embodiments of maytansinoid drug moieities include: DM1; DM3;and DM4, having the structures:

wherein the wavy line indicates the covalent attachment of the sulfuratom of the drug to a linker (L) of an antibody drug conjugate.HERCEPTIN® (trastuzumab, anti-HER 2 antibody) linked by SMCC to DM1 hasbeen reported (WO 2005/037992, which is expressly incorporated herein byreference in its entirety). An antibody drug conjugate of the presentinvention may be prepared according to the procedures disclosed therein.

Other exemplary maytansinoid antibody drug conjugates have the followingstructures and abbreviations, (wherein Ab is antibody and p is 1 toabout 8):

Exemplary antibody drug conjugates where DM1 is linked through a BMPEOlinker to a thiol group of the antibody have the structure andabbreviation:

where Ab is antibody; n is 0, 1, or 2; and p is 1, 2, 3, or 4.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×10⁵ HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Anti-CD22 antibody-maytansinoid conjugates are prepared by chemicallylinking an antibody to a maytansinoid molecule without significantlydiminishing the biological activity of either the antibody or themaytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020 (thedisclosure of which is hereby expressly incorporated by reference). Anaverage of 3-4 maytansinoid molecules conjugated per antibody moleculehas shown efficacy in enhancing cytotoxicity of target cells withoutnegatively affecting the function or solubility of the antibody,although even one molecule of toxin/antibody would be expected toenhance cytotoxicity over the use of naked antibody. Maytansinoids arewell known in the art and can be synthesized by known techniques orisolated from natural sources. Suitable maytansinoids are disclosed, forexample, in U.S. Pat. No. 5,208,020 and in the other patents andnonpatent publications referred to hereinabove. Preferred maytansinoidsare maytansinol and maytansinol analogues modified in the aromatic ringor at other positions of the maytansinol molecule, such as variousmaytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. Nos. 5,208,020, 6,441,163, or EP Patent 0 425 235B1, Chari et al., Cancer Research 52:127-131 (1992), and US 2005/0169933A1, the disclosures of which are hereby expressly incorporated byreference. Antibody-maytansinoid conjugates comprising the linkercomponent SMCC may be prepared as disclosed in U.S. patent applicationSer. No. 11/141,344, filed 31 May 2005, “Antibody Drug Conjugates andMethods”. The linking groups include disulfide groups, thioether groups,acid labile groups, photolabile groups, peptidase labile groups, oresterase labile groups, as disclosed in the above-identified patents.Additional linking groups are described and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

In one embodiment, any of the antibodies of the invention (full lengthor fragment) is conjugated to one or more maytansinoid molecules. In oneembodiment of the immunoconjugate, the cytotoxic agent D, is amaytansinoid DM1. In one embodiment of the immunoconjugate, the linkeris SMCC. In one embodiment, the antibody-linker-drug conjugate is ananti-CD22 antibody as disclosed herein to which is covalently DM1cytotoxic agent via the SMCC linker.

Auristatins and Dolostatins

In some embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in “Senter etal, Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented Mar. 28, 2004, the disclosure ofwhich is expressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE (wherein the wavy lineindicates the covalent attachment to a linker (L) of an antibody drugconjugate).

Another exemplary auristatin embodiment is MMAF, wherein the wavy lineindicates the covalent attachment to a linker (L) of an antibody drugconjugate (US 2005/0238649):

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 8):

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Liibke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol21(7):778-784.

Calicheamicin

In other embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ₁^(I) (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al.,Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patentsto American Cyanamid). Another anti-tumor drug that the antibody can beconjugated is QFA which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SLAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

Preparation of Antibody Drug Conjugates:

In the antibody drug conjugates (ADC) of the invention, an antibody (Ab)is conjugated to one or more drug moieties (D), e.g. about 1 to about 20drug moieties per antibody, through a linker (L). The ADC of Formula Imay be prepared by several routes, employing organic chemistryreactions, conditions, and reagents known to those skilled in the art,including: (1) reaction of a nucleophilic group of an antibody with abivalent linker reagent, to form Ab-L, via a covalent bond, followed byreaction with a drug moiety D; and (2) reaction of a nucleophilic groupof a drug moiety with a bivalent linker reagent, to form D-L, via acovalent bond, followed by reaction with the nucleophilic group of anantibody. Additional methods for preparing ADC are described herein.

Ab-(L-D)_(p)  Formula I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio)pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Exemplary linker component structures are shown below (wherein the wavyline indicates sites of covalent attachment to other components of theADC):

Additional exemplary linker components and abbreviations include(wherein the antibody (Ab) and linker are depicted, and p is 1 to about8):

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g. withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g. by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either glactose oxidase or sodium meta-periodate may yieldcarbonyl (aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

In yet another aspect, the antibody has one or more lysine residues thatcan be chemically modified to introduce one or more sulfhydryl groups.The antibody unit bonds to the Linker unit via the sulfhydryl group'ssulfur atom. The reagents that can be used to modify lysines include,but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and2-Iminothiolane hydrochloride (Traut's Reagent).

In another embodiment, the antibody can have one or more carbohydrategroups that can be chemically modified to have one or more sulfhydrylgroups. The antibody unit bonds to the Linker Unit, such as theStretcher Unit, via the sulfhydryl group's sulfur atom, as disclosedherein.

In yet another embodiment, the antibody can have one or morecarbohydrate groups that can be oxidized to provide an aldehyde (—CHO)group (see, for e.g., Laguzza, et al., J. Med. Chem. 1989, 32(3),548-55). The corresponding aldehyde can form a bond with a Reactive Siteon a Stretcher. Reactive sites on a Stretcher that can react with acarbonyl group on an antibody include, but are not limited to, hydrazineand hydroxylamine. Other protocols for the modification of proteins forthe attachment or association of Drug Units are described in Coligan etal., Current Protocols in Protein Science, vol. 2, John Wiley & Sons(2002), incorporated herein by reference.

Methods for the conjugation of linker-drug moieties to cell-targetedproteins such as antibodies, immunoglobulins or fragments thereof arefound, for example, in U.S. Pat. No. 5,208,020; U.S. Pat. No. 6,441,163;WO2005037992; WO2005081711; and WO2006/034488, all of which are herebyexpressly incorporated by reference in their entirety.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

In one embodiment of the immunoconjugate, the cytotoxic agent, D, is anauristatin of formula D_(E) or D_(F)

and wherein R² and R⁶ are each methyl, R³ and R⁴ are each isopropyl, R⁷is sec-butyl, each R⁸ is independently selected from CH₃, O—CH₃, OH, andH; R⁹ is H; R¹⁰ is aryl; Z is —O— or —NH—; R¹¹ is H, C₁-C₈ alkyl, or—(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₃; and R¹⁸ is —C(R⁸)₂—C(R⁸)₂-aryl; and

(d) p ranges from about 1 to 8.

The following embodiments are further provided for any of the aboveimmunoconjugates. In one embodiment, an immunoconjugate has in vitro orin vivo cell killing activity. In one embodiment, the linker is attachedto the antibody through a thiol group on the antibody. In oneembodiment, the linker is cleavable by a protease. In one embodiment,the linker comprises a val-cit dipeptide. In one embodiment, the linkercomprises a p-aminobenzyl unit. In one embodiment, the p-aminobenzylunit is disposed between the drug and a protease cleavage site in thelinker. In one embodiment, the p-aminobenzyl unit isp-aminobenzyloxycarbonyl (PAB). In one embodiment, the linker comprises6-maleimidocaproyl. In one embodiment, the 6-maleimidocaproyl isdisposed between the antibody and a protease cleavage site in thelinker. The above embodiments may occur singly or in any combinationwith one another.

In one embodiment, the drug is selected from MMAE and MMAF. In oneembodiment, the immunoconjugate has the formula

wherein Ab is any of the above anti-CD22 antibodies, S is a sulfur atom,and p ranges from 2 to 5. In one embodiment, the immunoconjugate has theformula

wherein Ab is any of the above anti-CD22 antibodies, S is a sulfur atom,and p ranges from about 1 to about 6, from about 2 to about 5, fromabout 2 to about 6, from about 2 to about 4, from about 2 to about 3,from about 3 to about 4, from about 3 to about 5, from about 3 to about6, or from about 4 to about 6.

Labelled Antibody Imaging Methods:

In another embodiment of the invention, cysteine engineered antibodiesmay be labelled through the cysteine thiol with radionuclides,fluorescent dyes, bioluminescence-triggering substrate moieties,chemiluminescence-triggering substrate moieties, enzymes, and otherdetection labels for imaging experiments with diagnostic,pharmacodynamic, and therapeutic applications. Generally, the labelledcysteine engineered antibody, i.e. “biomarker” or “probe”, isadministered by injection, perfusion, or oral ingestion to a livingorganism, e.g. human, rodent, or other small animal, a perfused organ,or tissue sample. The distribution of the probe is detected over a timecourse and represented by an image.

Articles of Manufacture:

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the disordersdescribed above is provided. The article of manufacture comprises acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, blister pack, etc. The containers may be formed from a varietyof materials such as glass or plastic. The container holds anantibody-drug conjugate (ADC) composition which is effective fortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is an ADC. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

Pharmaceutical Compositions:

In one aspect, a pharmaceutical composition is provided comprising anyof the above immunoconjugates and a pharmaceutically acceptable carrier.In one aspect, a method of treating a B cell proliferative disorder isprovided, wherein the method comprises administering to an individualthe pharmaceutical composition. In one embodiment, the B cellproliferative disorder is selected from lymphoma, non-Hodgkins lymphoma(NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL),acute lymphocytic leukemia (ALL), and mantle cell lymphoma. In oneembodiment, the cell proliferative disorder is associated with increasedexpression of CD22 on the surface of a cell.

In one aspect, a method of inhibiting cell proliferation is provided,wherein the method comprises exposing a cell to any of the aboveimmunoconjugates under conditions permissive for binding of theimmunoconjugate to CD22. In one embodiment, the B cell is a tumor cell.In one embodiment, the tumor cell is a B cell of a mammal experiencingor suspected of experiencing a B cell proliferative disorder selectedfrom lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsedaggressive NHL, relapsed indolent NHL, refractory NHL, refractoryindolent NHL, chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocyticleukemia (ALL), and mantle cell lymphoma., the cell is a xenograft. Inone embodiment, the exposing takes place in vitro. In one embodiment,the exposing takes place in vivo.

In one aspect, a method of using the anti-CD22 antibody of the inventionis provided to assay serum soluble CD22 in a mammal experiencingleukemia or lymphoma to diagnose B-cell leukemia or B-cell lymphoma,measuring clinical progression or regression of the diseases, or assesstumor burden or relapse. Such methods are disclosed in US 20050244828(Kreitman, R. J. et al., the entire contents of which is herebyincorporated by reference) using an anti-CD22 RFB4 antibody PE38(Pseudomonas exotoxin A fragment 38) toxin conjugate (see Kreitman, R.J. et al., NEJM 345:241-247 (2001)).

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D: FIG. 1A is a diagram of CD22 indicating the sevenimmunoglobulin-like domains of the extracellular domain of the betaisoform. The alpha isoform lacks domains 3 and 4. “TM” refers totransmembrane domain. FIG. 1B depicts the amino acid sequence of thebeta form of CD22 (SEQ ID NO:27). The alpha form of CD22 lacks the aminoacids shown in italics (encoding domains 3 and 4 of the extracellulardomain). The extracellular domain of the mature form of the protein isunderlined (SEQ ID NO:28). Amino acids 1-21 depict the signal sequencecleaved from the mature form. FIG. 1C is the amino acid sequence ofCD22alpha (SEQ ID NO:29). The ECD of CD22alpha is underlined (SEQ IDNO:30). FIG. 1D is the amino acid sequence of CD22 from cynomolgusmonkey (cyno) (SEQ ID NO:31). The first 19 amino acids of cyno CD22 isthe signal sequence.

FIGS. 2A-2B: FIG. 2A depicts the amino acid sequence of the heavy chainvariable region of murine 10F4 anti-CD22 antibody of the invention(m10F4) aligned with the humanized 10F4 version 1 antibody (h10F4v1) andaligned with the human subgroup III sequence. The HVRs are boxed(HVR-H1, HVR-H2, HVR-H3). The sequences bracketing the HVRs are theframework sequences (FR-H1 to FR-H4). The sequences are numberedaccording to Kabat numbering. The Kabat, Chothia, and contact CDRs areindicated about the boxed HVRs. FIG. 2B depicts the amino acid sequenceof the light chain variable region of murine 10F4 anti-CD22 antibody ofthe invention (m10F4) aligned with the humanized 10F4 version 1 antibody(h10F4v1) and aligned with the human kappa I sequence. Versions 2 and 3of the humanized 10F4 antibody (h10F4v2 and h10F4v3) have the same aminoacid sequences for the secreted mature form. The antibodies h10F4v2 andh10F4v3 differ from h10F4v1 at amino acid 28 of the HVR-L1 (N28V). TheHVRs are boxed. The FR-L1, FR-L2, FR-L3, and FR-L4 sequences bracket theHVRs (HVR-L1, HVR-L2, HVR-L3). The sequences are numbered according toKabat numbering. The Kabat, Chothia, and contact CDRs are indicatedabout the boxed HVRs.

FIGS. 3A and 3B show exemplary acceptor human variable heavy (VH)consensus framework sequences for use in practicing the instantinvention with sequence identifiers as follows, where the FR SEQ ID NOsare listed in the order FR-H1, FR-H2, FR-H3, FR-H4:

-   -   human VH subgroup I consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:26, 47, 48, 7).    -   human VH subgroup I consensus frameworks “B,” “C,” and “D” minus        extended hypervariable regions (SEQ ID NOs:50, 51, 52, 7; SEQ ID        NOs:50, 51, 52, 7; and SEQ ID NOs:50, 51, 53, 7).    -   human VH subgroup II consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:54, 55, 56, 7).    -   human VH subgroup II consensus frameworks “B,” “C,” and “D”        minus extended hypervariable regions (SEQ ID NOs:57, 58, 56, 7;        SEQ ID NOs:57, 58, 59, 7; and SEQ ID NOs:57, 58, 60, 7).    -   human VH subgroup III consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:61, 62, 63, 7).    -   human VH subgroup III consensus frameworks “B,” “C,” and “D”        minus extended hypervariable regions (SEQ ID NOs:64, 65, 63, 7;        SEQ ID NOs:64, 65, 66, 7; and SEQ ID NOs:64, 65, 67, 7).    -   human VH acceptor 1 framework “A” minus Kabat CDRs (SEQ ID        NOs:68, 62, 69, 7).    -   human VH acceptor frameworks “B” and “C” minus extended        hypervariable regions (SEQ ID NOs:64, 65, 69, 7; and SEQ ID        NOs:64, 65, 70, 7).    -   human VH acceptor 2 framework “A” minus Kabat CDRs (SEQ ID        NOs:68, 62, 71, 7).    -   human VH acceptor 2 framework “B,” “C,” and “D” minus extended        hypervariable regions (SEQ ID NOs:64, 65, 71, 7; SEQ ID NOs:64,        65, 72, 7; and SEQ ID NOs:64, 65, 73, 7).

FIGS. 4A and 4B show exemplary acceptor human variable light (VL)consensus framework sequences for use in practicing the instantinvention with sequence identifiers as follows:

-   -   human VL kappa subgroup I-1 consensus framework (κv1-1): SEQ ID        NOs:74, 75, 76, 77    -   human VL kappa subgroup I consensus framework (κv1): SEQ ID        NOs:74, 78, 76, 77    -   human VL kappa subgroup II consensus framework (κv2): SEQ ID        NOs:49, 79, 80, 77    -   human VL kappa subgroup III consensus framework (κv3): SEQ ID        NOs:81, 82, 83, 77    -   human VL kappa subgroup IV consensus framework (κv4): SEQ ID        NOs:84, 85, 86, 77

FIGS. 5A and 5B: FIG. 5A depicts alignments of native sequence human IgGFc region sequences, humIgG1 (non-A allotype, SEQ ID NO:38; and Aallotype, where the amino acid sequence SREEM within SEQ ID NO:38 ischanged to SRDEL), humIgG2 (SEQ ID NO:39), humIgG3 (SEQ ID NO:40) andhumIgG4 (SEQ ID NO:41) with differences between the sequences markedwith asterisks. Numbers above the sequences represent the EU numberingsystem. An exemplary kappa constant region is also shown. FIG. 5Bdepicts the full length amino acid sequences (variable and constantregions) of the light and heavy chains of humanized anti-CD22 antibody10F4v2, isotype IgG1. The underlined portions depict the constantdomains.

FIGS. 6A-6D show results of assays measuring various determinants ofCD22 ADC efficacy in lymphoma cell lines. FIG. 6A indicates that highercell surface CD22 levels are correlated to a lower anti-CD22-MCC-DM1IC50 (higher efficacy). FIG. 6B indicates that increased internalizationof anti-CD22-MCC-DM1 correlates with lower anti-CD22-MCC-DM1 IC50. FIG.6C indicates that increased intrinsic sensitivity of cells to free drugcorrelates with lower anti-CD22-MCC-DM1 IC50. FIG. 6D is aphotomicrograph showing the internalization of fluorescently labeledanti-CD22 antibody following binding to CD22 on the cell surface.

FIGS. 7A-7B: FIG. 7A is a graph of in vivo tumor volume reduction in axenograft model which shows that administration of anti-CD22 antibodymu10F4-smcc-DM1 and hu10F4v1-smcc-DM1 to SCID mice having human B celltumors significantly reduced tumor volume. Drug load was approximately 4and 4.6, see Table 4. FIG. 7B is a graph of a similar study, but drugload was slightly lower at approximately 2.9 and 3.0 (see Table 5), andmu10F4-smcc-DM1 and hu10F4v2-smcc-DM1 efficacy were compared withcontrol antibody and unconjugated mu10F4. FIG. 7C is a graph of in vivotumor reduction in a xenograft model in which anti-CD22-spp-DM1 wasadministered as indicated in Table 6.

FIGS. 8A and 8B: FIG. 8A is a graph of anti-CD22 antibodies5E8.1.8-smcc-DM1 and RFB4-smcc-DM1 administered to Ramos cellxenografts. FIG. 8B is a graph of anti-CD22 antibodies 5E8.1.8-smcc-DM1and RFB4-smcc-DM1 administered to BJAB-luc xenografts.

FIG. 9 is a graph showing the relative affect on tumor volume over timeafter administration of anti-CD22(RFB4)-smcc-DM1 at low, medium, andhigh drug loads.

FIG. 10 is a graph showing the relative affect on tumor volume over timeafter administration of anti-CD22(RFB4)-MC-vcPAB-MMAF oranti-CD22(RFB4)-MC-MMAF in Ramos xenografts.

FIG. 11 is a graph showing the relative affect on tumor volume over timeafter administration of anti-CD22(RFB4)-smcc-DM1 or -MCvcPAB-MMAE.

FIG. 12 is a graph showing the relative affect on tumor volume over timeafter administration of humanized anti-CD22 10F4 variants as MMAF or DM1immunoconjugates as disclosed in Table 12.

FIGS. 13A-13C are graphs showing the relative affect on tumor volumeover time after administration anti-CD22-smcc-DM1 or anti-CD22-MC-MMAFin different B cell lymphoma xenograft models: SuDHL-4 (FIG. 13A), DoHH2(FIG. 13B), and Granta-519 (FIG. 13C).

FIG. 14 shows diagrams of CD22 domains deleted for epitope mapping asdescribed in the Examples. The domains are numbered 1-7. “TM” refers totransmembrane domain.

FIG. 15 shows depictions of cysteine engineered anti-CD22 antibody drugconjugates (ADC) where a drug moiety is attached to an engineeredcysteine group in: the light chain (LC-ADC); the heavy chain (HC-ADC);and the Fc region (Fc-ADC).

FIG. 16 shows the steps of: (i) reducing cysteine disulfide adducts andinterchain and intrachain disulfides in a cysteine engineered anti-CD22antibody (ThioMab) with reducing agent TCEP(tris(2-carboxyethyl)phosphine hydrochloride); (ii) partially oxidizing,i.e. reoxidation to reform interchain and intrachain disulfides, withdhAA (dehydroascorbic acid); and (iii) conjugation of the reoxidizedantibody with a drug-linker intermediate to form a cysteine engineeredanti-CD22 antibody drug conjugate (ADC).

FIGS. 17A-17C depict the amino acid sequences of the anti-CD22 cysteineengineered antibodies of the invention in which the light chain or heavychain or Fc region is altered to engineer a cysteine at selected aminoacid positions. FIG. 17A depicts the amino acid sequence of theanti-CD22 10F4 variant light chain in which a valine at Kabat position205 (sequential position Valine 210) is altered to a Cysteine. FIG. 17Bdepicts the amino acid sequence of the anti-CD22 10F4 variant heavychain in which an Alanine at EU position 118 (sequential positionAlanine 121) is altered to a Cysteine. FIG. 17C depicts the amino acidsequence of the anti-CD22 10F4 variant Fc region in which a Serine at EUposition 400 (sequential position Serine 403) is altered to a Cysteine.In each figure, the altered amino acid is shown in bold text with doubleunderlining. Single underlining indicates constant regions. Variableregions are not underlined.

FIGS. 18A-18E are FACS plots indicating that binding of anti-CD22thiomab drug conjugates (TDCs) of the invention bind to CD22 expressedon the surface of BJAB-lucs cells is similar for LC, HC and Fc thiomabvariants as well as for the different drug conjugates shown.

FIG. 19 is a graph plotting changes in mean tumor volume over time in axenograft model treated with different anti-CD22 TDCs, which varied byposition of the engineered cysteine (LC, HC or Fc) and/or by drugconjugate (MMAF or MMAE). Xenograft models treated with anti-CD22 TDCs10F4-LC-V210C-MCvcPAB-MMAE and anti-CD22 10F4-HC-A121C-MCvcPAB-MMAEshowed a decrease in tumor volume during the study.

FIG. 20A is a graph plotting changes in mean tumor volume over time in ahuman mantle cell lymphoma Granta-519 xenograft in CB17 SCID micetreated with heavy chain A118C anti-CD22 TDCs conjugated to differentlinker drug moieties and/or administered at different doses as shown.The anti-CD22 10F4-HC(A118C)-MCvcPAB-MMAE TDC appeared to be the mostefficacious of the test agents in this experiment.

FIG. 20B is a graph plotting changes in mean tumor volume over time in afollicular lymphoma DOHH2 xenograft in CB17 SCID mice treated with thesame heavy chain A118C anti-CD22 TDCs, but at higher doses. Theanti-CD22 10F4-HC(A118C)-MCvcPAB-MMAE TDC appeared to be the mostefficacious of the test agents in this experiment. FIG. 20C is a plot ofpercent weight change is the mice from the DOHH2 xenograft study showingthat there was no significant change in weight during the first 14 daysof the study.

FIGS. 21A and 21B are bar graphs showing changes in serum AST (aspartateaminotransferase) (FIG. 21A) and serum neutrophils (FIG. 21B) at Days 0and 5 where ADC comprising a cleavable and uncleavable linker wasadministered.

FIGS. 22A and 22B are graphs showing depletion of peripheral B cells(CD20⁺ cells) in cynomolgus monkeys dosed with 10, 20, and 30 mg/kganti-CD22 MMAF (FIG. 22A) and anti-CD22 DM1 (FIG. 22B).

FIGS. 23A and 23B are graphs showing no significant change in CD4⁺lymphocytes at 10, 20, and 30 mg/kg anti-CD22 MMAF (FIG. 23A) andanti-CD22 DM1 (FIG. 23B).

FIGS. 24A and 24B show histological samples of cynomolgus monkey tonsiltissue in which depletion of germinal center B cells, apparent in thevehicle control (FIG. 24A), are depleted in a tonsil sample from ananimal dosed at 10 mg/kg hu10F4v3-SMCC-DM1.

FIG. 25A is a diagram indicating the regions of spleen follicle fromwhich tissue samples were taken for a study in which it was shown thatanti-CD22 ADCs spare B cells in resting tissue in cynomolgus monkeys.Dividing cells in cyno spleen follicle germinal center were depleted individing germinal cells of cyno spleens from animals dosed withhu10F4v3-MC-MMAF at 10 mg/kg (FIGS. 25B and 25C). Non-dividing naïve Bcells were not depleted under the same conditions (FIGS. 25D and 25E).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Isolated antibodies that bind to CD22 are provided. Immunoconjugatescomprising anti-CD22 antibodies are further provided. Cysteineengineered anti-CD22 antibodies and immunoconjugates thereof are furtherprovided. Antibodies and immunoconjugates of the invention are useful,e.g., for the diagnosis or treatment of disorders associated withaltered expression, e.g., increased expression, of CD22. In certainembodiments, antibodies or immunoconjugates of the invention are usefulfor the diagnosis or treatment of a cell proliferative disorder, such asa tumor or cancer. In certain embodiments, antibodies orimmunoconjugates of the invention are useful for the detection of CD22,e.g., CD22 expressed on the cell surface.

Polynucleotides encoding anti-CD22 antibodies are provided. Vectorscomprising polynucleotides encoding anti-CD22 antibodies are provided,and host cells comprising such vectors are provided. Compositions,including pharmaceutical formulations, comprising any one or more of thepolynucleotides, anti-CD22 antibodies, or immunoconjugates of theinvention are also provided.

General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): Pcr 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J.B. LippincottCompany, 1993).

DEFINITIONS AND ABBREVIATIONS Definitions

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, an antibody is purified(1) to greater than 95% by weight of antibody as determined by, forexample, the Lowry method, and in some embodiments, to greater than 99%by weight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isseparated from at least one other nucleic acid molecule with which it isordinarily associated, for example, in its natural environment. Anisolated nucleic acid molecule further includes a nucleic acid moleculecontained in cells that ordinarily express the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

“Purified” means that a molecule is present in a sample at aconcentration of at least 95% by weight, or at least 98% by weight ofthe sample in which it is contained.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may comprise modification(s)made after synthesis, such as conjugation to a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO, or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle-stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

A “B-cell surface marker” or “B-cell surface antigen” herein is anantigen expressed on the surface of a B cell that can be targeted withan antagonist that binds thereto, including but not limited to,antibodies to a B-cell surface antigen or a soluble form a B-cellsurface antigen capable of antagonizing binding of a ligand to thenaturally occurring B-cell antigen. Exemplary B-cell surface markersinclude the CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53,CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81,CD82, CD83, CDw84, CD85 and CD86 leukocyte surface markers (fordescriptions, see The Leukocyte Antigen Facts Book, 2^(nd) Edition.1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., NewYork). Other B-cell surface markers include RP105, FcRH2, B-cell CR2,CCR6, P2×5, HLA-DOB, CXCR5, FCER2, BR3, BAFF, BLyS, Btig, NAG14,SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287.The B-cell surface marker of particular interest is preferentiallyexpressed on B cells compared to other non-B-cell tissues of a mammaland may be expressed on both precursor B cells and mature B cells.

The term “CD22,” as used herein, refers to any native CD22 from anyvertebrate source, including mammals such as primates (e.g. humans,cynomolgus monkey (cyno)) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessedCD22 as well as any form of CD22 that results from processing in thecell. The term also encompasses naturally occurring variants of CD22,e.g., splice variants, allelic variants, and isoforms. The major isoformof CD22 (CD22beta) comprises 847 amino acids and sevenimmunoglobulin-like regions in the extracellular domain (see Wilson, G.L. et al., J. Exp. Med. 173:137-146 (1991)). A minor isoform, CD22alpha,comprises 647 amino acids and lacks immunoglobulin-like domains 3 and 4in the extracellular domain (see Stamenkovic, I. and Seed, B., Nature345:74-77 (1990)) and Wilson et al. (1991), supra). The amino acidsequence of CD22 beta is depicted in FIG. 1B in which the underlinedportion is the extracellular domain (ECD) and the italicized portionindicates the amino acids missing from the CD22 alpha extracellulardomain sequence. FIG. 1C depicts the amino acid sequence of CD22alpha inwhich the ECD is underlined. The amino acid sequence from amino acid 1to amino acid 21 represents the signal sequence cleaved from the matureform of the protein. In one embodiment, CD22 is expressed on the cellsurface, such as on the surface of a normal B cell or a tumor B cell.FIG. 1D depicts the amino acid sequence of CD22 from cynomolgus monkey.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingsimilar structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which generally lackantigen specificity. Polypeptides of the latter kind are, for example,produced at low levels by the lymph system and at increased levels bymyelomas.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full lengthor intact monoclonal antibodies), polyclonal antibodies, monovalentantibodies, multivalent antibodies, multispecific antibodies (e.g.,bispecific antibodies so long as they exhibit the desired biologicalactivity) and may also include certain antibody fragments (as describedin greater detail herein). An antibody can be chimeric, human, humanizedand/or affinity matured.

The term “anti-CD22 antibody” or “an antibody that binds to CD22” refersto an antibody that is capable of binding CD22 with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting CD22. Preferably, the extent of binding of ananti-CD22 antibody to an unrelated, non-CD22 protein is less than about10% of the binding of the antibody to CD22 as measured, e.g., by aradioimmunoassay (RIA). In certain embodiments, an antibody that bindsto CD22 has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1nM, or ≦0.1 nM. In certain embodiments, an anti-CD22 antibody binds toan epitope of CD22 that is conserved among CD22 from different species.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions (HVRs) both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md. (1991)). The constant domains are notinvolved directly in the binding of an antibody to an antigen, butexhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (x) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibodymay be part of a larger fusion molecule, formed by covalent ornon-covalent association of the antibody with one or more other proteinsor peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containthe Fc region.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion retains at least one, and as many as most or all, ofthe functions normally associated with that portion when present in anintact antibody. In one embodiment, an antibody fragment comprises anantigen binding site of the intact antibody and thus retains the abilityto bind antigen. In another embodiment, an antibody fragment, forexample one that comprises the Fc region, retains at least one of thebiological functions normally associated with the Fc region when presentin an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise on antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO93/1161; Hudson et al. (2003) Nat. Med. 9:129-134; andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al. (2003)Nat. Med. 9:129-134.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2^(nd) ed. 1988); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567), phage display technologies(see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al.,J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004);Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); andLee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), andtechnologies for producing human or human-like antibodies in animalsthat have parts or all of the human immunoglobulin loci or genesencoding human immunoglobulin sequences (see, e.g., WO98/24893;WO96/34096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad.Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Markset al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al.,Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93(1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit, or nonhuman primate having the desired specificity,affinity, and/or capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH(H1, H2, H3), andthree in the VL (L1, L2, L3). In native antibodies, H3 and L3 displaythe most diversity of the six hypervariable regions, and H3 inparticular is believed to play a unique role in conferring finespecificity to antibodies. Xu et al. (2000) Immunity 13:37-45; Johnsonand Wu (2003) in Methods in Molecular Biology 248:1-25 (Lo, ed., HumanPress, Totowa, N.J.). Indeed, naturally occurring camelid antibodiesconsisting of a heavy chain only are functional and stable in theabsence of light chain. Hamers-Casterman et al. (1993) Nature363:446-448; Sheriff et al. (1996) Nature Struct. Biol. 3:733-736.

A number of hypervariable region delineations are in use and areencompassed herein. The Kabat Complementarity Determining Regions (CDRs)are based on sequence variability and are the most commonly used (Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)).Chothia refers instead to the location of the structural loops (Chothiaand Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM hypervariableregions represent a compromise between the Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular's AbM antibodymodeling software. The “contact” hypervariable regions are based on ananalysis of the available complex crystal structures. The residues fromeach of these hypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96(L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102,or 95-102 (H3) in the VH. The variable domain residues are numberedaccording to Kabat et al., supra, for each of these definitions. TheHVR-H1 and HVR-H2 hypervariable regions of the anti-CD22 10F4 antibodiesof the invention are H26-H35 and H49-H65 using Kabat numbering.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Using this numbering system, the actual linear amino acid sequence maycontain fewer or additional amino acids corresponding to a shorteningof, or insertion into, a FR or HVR of the variable domain. For example,a heavy chain variable domain may include a single amino acid insert(residue 52a according to Kabat) after residue 52 of H2 and insertedresidues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat)after heavy chain FR residue 82. The Kabat numbering of residues may bedetermined for a given antibody by alignment at regions of homology ofthe sequence of the antibody with a “standard” Kabat numbered sequence.

A “free cysteine amino acid” refers to a cysteine amino acid residuewhich has been engineered into a parent antibody, has a thiol functionalgroup (—SH), and is not paired as, or otherwise part of, anintramolecular or intermolecular disulfide bridge.

The term “thiol reactivity value” is a quantitative characterization ofthe reactivity of free cysteine amino acids. The thiol reactivity valueis the percentage of a free cysteine amino acid in a cysteine engineeredantibody which reacts with a thiol-reactive reagent, and converted to amaximum value of 1. For example, a free cysteine amino acid on acysteine engineered antibody which reacts in 100% yield with athiol-reactive reagent, such as a biotin-maleimide reagent, to form abiotin-labelled antibody has a thiol reactivity value of 1.0. Anothercysteine amino acid engineered into the same or different parentantibody which reacts in 80% yield with a thiol-reactive reagent has athiol reactivity value of 0.8. Another cysteine amino acid engineeredinto the same or different parent antibody which fails totally to reactwith a thiol-reactive reagent has a thiol reactivity value of 0.Determination of the thiol reactivity value of a particular cysteine maybe conducted by ELISA assay, mass spectroscopy, liquid chromatography,autoradiography, or other quantitative analytical tests. Thiol-reactivereagents which allow capture of the cysteine engineered antibody andcomparison and quantitation of the cysteine reactivity includebiotin-PEO-maleimide((+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctainediamine, Oda etal (2001) Nature Biotechnology 19:379-382, Pierce Biotechnology, Inc.)Biotin-BMCC, PEO-Iodoacetyl Biotin, Iodoacetyl-LC-Biotin, andBiotin-HPDP (Pierce Biotechnology, Inc.), andNα-(3-maleimidylpropionyl)biocytin (MPB, Molecular Probes, Eugene,Oreg.). Other commercial sources for biotinylation, bifunctional andmultifunctional linker reagents include Molecular Probes, Eugene, Oreg.,and Sigma, St. Louis, Mo.

A “parent antibody” is an antibody comprising an amino acid sequencefrom which one or more amino acid residues are replaced by one or morecysteine residues. The parent antibody may comprise a native or wildtype sequence. The parent antibody may have pre-existing amino acidsequence modifications (such as additions, deletions and/orsubstitutions) relative to other native, wild type, or modified forms ofan antibody. A parent antibody may be directed against a target antigenof interest, e.g. a biologically important polypeptide. Antibodiesdirected against nonpolypeptide antigens (such as tumor-associatedglycolipid antigens; see U.S. Pat. No. 5,091,178) are also contemplated.

The following abbreviations are used herein and have the indicateddefinitions: BME is beta-mercaptoethanol, Boc is N-(t-butoxycarbonyl),cit is citrulline (2-amino-5-ureido pentanoic acid), dap is dolaproine,DCC is 1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA isdiethylamine, DEAD is diethylazodicarboxylate, DEPC isdiethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA isN,N-diisopropylethylamine, dil is dolaisoleucine, DMA isdimethylacetamide, DMAP is 4 DME is ethyleneglycol dimethyl ether (or1,2-dimethoxyethane), DMF is N,N-dimethylformamide, DMSO isdimethylsulfoxide, doe is dolaphenine, dov is N,N-dimethylvaline, DTNBis 5,5′-dithiobis(2-nitrobenzoic acid), DTPA isdiethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospraymass spectrometry, EtOAc is ethyl acetate, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HOBt is 1 HPLC is high pressure liquidchromatography, ile is isoleucine, lys is lysine, MeCN(CH₃CN) isacetonitrile, MeOH is methanol, Mtr is 4-anisyldiphenylmethyl (or4-methoxytrityl), nor is (1S,2R)-(+)-norephedrine, PAB isp-aminobenzylcarbamoyl, PBS is phosphate-buffered saline (pH 7), PEG ispolyethylene glycol, Ph is phenyl, Pnp is p-nitrophenyl, MC is6-maleimidocaproyl, phe is L-phenylalanine, PyBrop is bromotris-pyrrolidino phosphonium hexafluorophosphate, SEC is size-exclusionchromatography, Su is succinimide, TFA is trifluoroacetic acid, TLC isthin layer chromatography, UV is ultraviolet, and val is valine.

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). In one embodiment, an affinity maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity matured antibodies are produced by procedures known inthe art. Marks et al. Bio/Technology 10:779-783 (1992) describesaffinity maturation by VH and VL domain shuffling. Random mutagenesis ofHVR and/or framework residues is described by: Barbas et al. Proc Nat.Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al.,J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “agonist antibody,” as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see Daeron, Annu.Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch andKinet, Annu Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41(1995). Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) and regulation of homeostasis ofimmunoglobulins. Methods of measuring binding to FcRn are known (see,e.g., Ghetie 1997, Hinton 2004). Binding to human FcRn in vivo and serumhalf life of human FcRn high affinity binding polypeptides can beassayed, e.g., in transgenic mice or transfected human cell linesexpressing human FcRn, or in primates administered with the Fc variantpolypeptides.

WO00/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. The content of that patent publication isspecifically incorporated herein by reference. See, also, Shields et al.J. Biol. Chem. 9(2): 6591-6604 (2001).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function(s). Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils. The effector cells may be isolated from a native source,e.g., from blood.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The primary cells for mediatingADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarizedin Table 3 on page 464 of Ravetch and Kinet, Annu Rev. Immunol 9:457-92(1991). To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 or Presta U.S. Pat. No. 6,737,056 may be performed. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.PNAS (USA) 95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1 and WO99/51642. The contents of those patentpublications are specifically incorporated herein by reference. See,also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “Fc region-comprising polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin, which comprises an Fc region. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during purification of thepolypeptide or by recombinant engineering the nucleic acid encoding thepolypeptide. Accordingly, a composition comprising a polypeptide havingan Fc region according to this invention can comprise polypeptides withK447, with all K447 removed, or a mixture of polypeptides with andwithout the K447 residue.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a VL or VH framework derived froma human immunoglobulin framework or a human consensus framework. Anacceptor human framework “derived from” a human immunoglobulin frameworkor a human consensus framework may comprise the same amino acid sequencethereof, or it may contain pre-existing amino acid sequence changes. Insome embodiments, the number of pre-existing amino acid changes are 10or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less. Where pre-existing amino acid changes arepresent in a VH, preferably those changes occur at only three, two, orone of positions 71H, 73H and 78H; for instance, the amino acid residuesat those positions may be 71A, 73T and/or 78A. In one embodiment, the VLacceptor human framework is identical in sequence to the VL humanimmunoglobulin framework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, 5^(th)Ed. Public Health Service, National Institutes of Health, Bethesda, Md.(1991). In one embodiment, for the VL, the subgroup is subgroup kappa Ias in Kabat et al., supra. In one embodiment, for the VH, the subgroupis subgroup III as in Kabat et al., supra.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al., supra. In one embodiment, the VH subgroup III consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

EVQLVESGGGLVQPGGSLRLSCAAS (FR-H1, SEQ ID NO: 1)-HVR-H1-WVRQAPGKGLEWV (FR-H2, SEQ ID NO: 3)-HVR-H2-RFTISADTSKNTAYLQMNSLRAEDTAVYYC (FR-H3, SEQ ID NO: 5)-HVR-H3-WGQGTLVTVSS (FR-H4, SEQ ID NO: 7).

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al., supra. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

DIQMTQSPSSLSASVGDRVTITC (FR-L1, SEQ ID NO: 8)-HVR-L1-WYQQKPGKAPKLLIY (FR-L2, SEQ ID NO: 11)-HVR-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (FR-L3, SEQ ID NO: 13)-HVR-L3-FGQGTKVEIK (FR-L4, SEQ ID NO: 15).

“Secretion signal sequence” or “signal sequence” refers to a nucleicacid sequence encoding a short signal peptide that can be used to directa newly synthesized protein of interest through a cellular membrane,usually the inner membrane or both inner and outer membranes ofprokaryotes. As such, the protein of interest such as the immunoglobulinlight or heavy chain polypeptide is secreted into the periplasm of theprokaryotic host cells or into the culture medium. The signal peptideencoded by the secretion signal sequence may be endogenous to the hostcells, or they may be exogenous, including signal peptides native to thepolypeptide to be expressed. Secretion signal sequences are typicallypresent at the amino terminus of a polypeptide to be expressed, and aretypically removed enzymatically between biosynthesis and secretion ofthe polypeptide from the cytoplasm. Thus, the signal peptide is usuallynot present in a mature protein product.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay. Solution binding affinity of Fabs for antigen ismeasured by equilibrating Fab with a minimal concentration of(125I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol. 293:865-881).To establish conditions for the assay, microtiter plates (Dynex) arecoated overnight with 5 μg/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [¹²⁵I]-antigen antigen are mixed with serialdilutions of a Fab of interest (e.g., consistent with assessment of theanti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res.57:4593-4599). The Fab of interest is then incubated overnight; however,the incubation may continue for a longer period (e.g., about 65 hours)to ensure that equilibrium is reached. Thereafter, the mixtures aretransferred to the capture plate for incubation at room temperature(e.g., for one hour). The solution is then removed and the plate washedeight times with 0.1% Tween-20 in PBS. When the plates have dried, 150μl/well of scintillant (MicroScint-20; Packard) is added, and the platesare counted on a Topcount gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, the Kd or Kd value is measured by usingsurface plasmon resonance assays using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (kon) and dissociation rates (koff) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio koff/kon. See, e.g., Chen, Y.,et al., (1999) J. Mol. Biol. 293:865-881. If the on-rate exceeds 106M-1s-1 by the surface plasmon resonance assay above, then the on-ratecan be determined by using a fluorescent quenching technique thatmeasures the increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

An “on-rate,” “rate of association,” “association rate,” or “k_(on)”according to this invention can also be determined as described aboveusing a BIAcore™-2000 or a BIAcore™-3000 system (BIAcore, Inc.,Piscataway, N.J.).

A “disorder” is any condition or disease that would benefit fromtreatment with an substance/molecule or method of the invention. Thisincludes chronic and acute disorders including those pathologicalconditions which predispose the mammal to the disorder in question.Non-limiting examples of disorders to be treated herein includecancerous conditions such as B cell proliferative disorders and/or Bcell tumors, e.g., lymphoma, non-Hodgkins lymphoma (NHL), aggressiveNHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL,refractory indolent NHL, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acutelymphocytic leukemia (ALL), and mantle cell lymphoma.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, cancerous B cell proliferative disorders B cellproliferative disorder is selected from lymphoma, non-Hodgkins lymphoma(NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL),acute lymphocytic leukemia (ALL), and mantle cell lymphoma. Othercancers conditions included, for example, carcinoma, lymphoma (e.g.,Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia.More particular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, leukemia and other lymphoproliferative disorders, and varioustypes of head and neck cancer.

A “B-cell malignancy” herein includes non-Hodgkin's lymphoma (NHL),including low grade/follicular NHL, small lymphocytic (SL) NHL,intermediate grade/follicular NHL, intermediate grade diffuse NHL, highgrade immunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma,AIDS-related lymphoma, and Waldenstrom's Macroglobulinemia,non-Hodgkin's lymphoma (NHL), lymphocyte predominant Hodgkin's disease(LPHD), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia(CLL), indolent NHL including relapsed indolent NHL andrituximab-refractory indolent NHL; leukemia, including acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairycell leukemia, chronic myeloblastic leukemia; mantle cell lymphoma; andother hematologic malignancies. Such malignancies may be treated withantibodies directed against B-cell surface markers, such as CD22. Suchdiseases are contemplated herein to be treated by the administration ofan antibody directed against a B cell surface marker, such as CD22, andincludes the administration of an unconjugated (“naked”) antibody or anantibody conjugated to a cytotoxic agent as disclosed herein. Suchdiseases are also contemplated herein to be treated by combinationtherapy including an anti-CD22 antibody or anti-CD22 antibody drugconjugate of the invention in combination with another antibody orantibody drug conjugate, another cytoxic agent, radiation or othertreatment administered simultaneously or in series. In exemplarytreatment method of the invention, an anti-CD22 antibody of theinvention is administered in combination with an anti-CD20 antibody,immunoglobulin, or CD20 binding fragment thereof, either together orsequentially. The anti-CD20 antibody may be a naked antibody or anantibody drug conjugate. In an embodiment of the combination therapy,the anti-CD22 antibody is an antibody of the present invention and theanti-CD20 antibody is Rituxan® (rituximab).

The term “non-Hodgkin's lymphoma” or “NHL”, as used herein, refers to acancer of the lymphatic system other than Hodgkin's lymphomas. Hodgkin'slymphomas can generally be distinguished from non-Hodgkin's lymphomas bythe presence of Reed-Sternberg cells in Hodgkin's lymphomas and theabsence of said cells in non-Hodgkin's lymphomas. Examples ofnon-Hodgkin's lymphomas encompassed by the term as used herein includeany that would be identified as such by one skilled in the art (e.g., anoncologist or pathologist) in accordance with classification schemesknown in the art, such as the Revised European-American Lymphoma (REAL)scheme as described in Color Atlas of Clinical Hematology (3rd edition),A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Ltd.,2000). See, in particular, the lists in FIGS. 11.57, 11.58 and 11.59.More specific examples include, but are not limited to, relapsed orrefractory NHL, front line low grade NHL, Stage III/IV NHL, chemotherapyresistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, smalllymphocytic lymphoma, B cell chronic lymphocytic leukemia and/orprolymphocytic leukemia and/or small lymphocytic lymphoma, B-cellprolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma,lymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenicmarginal zone lymphoma, extranodal marginal zone—MALT lymphoma, nodalmarginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasmacell myeloma, low grade/follicular lymphoma, intermediategrade/follicular NHL, mantle cell lymphoma, follicle center lymphoma(follicular), intermediate grade diffuse NHL, diffuse large B-celllymphoma, aggressive NHL (including aggressive front-line NHL andaggressive relapsed NHL), NHL relapsing after or refractory toautologous stem cell transplantation, primary mediastinal large B-celllymphoma, primary effusion lymphoma, high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, Burkitt's lymphoma, precursor (peripheral) large granularlymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, skin(cutaneous) lymphomas, anaplastic large cell lymphoma, angiocentriclymphoma.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or organs or a co-segregateor manifestation thereof or resulting condition therefrom. In many ofthese autoimmune and inflammatory disorders, a number of clinical andlaboratory markers may exist, including, but not limited to,hypergammaglobulinemia, high levels of autoantibodies, antigen-antibodycomplex deposits in tissues, benefit from corticosteroid orimmunosuppressive treatments, and lymphoid cell aggregates in affectedtissues. Without being limited to any one theory regarding B-cellmediated autoimmune disease, it is believed that B cells demonstrate apathogenic effect in human autoimmune diseases through a multitude ofmechanistic pathways, including autoantibody production, immune complexformation, dendritic and T-cell activation, cytokine synthesis, directchemokine release, and providing a nidus for ectopic neo-lymphogenesis.Each of these pathways may participate to different degrees in thepathology of autoimmune diseases.

“Autoimmune disease” can be an organ-specific disease (i.e., the immuneresponse is specifically directed against an organ system such as theendocrine system, the hematopoietic system, the skin, thecardiopulmonary system, the gastrointestinal and liver systems, therenal system, the thyroid, the ears, the neuromuscular system, thecentral nervous system, etc.) or a systemic disease which can affectmultiple organ systems (for example, systemic lupus erythematosus (SLE),rheumatoid arthritis, polymyositis, etc.). Preferred such diseasesinclude autoimmune rheumatologic disorders (such as, for example,rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupus such as SLEand lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia,anti-phospholipid antibody syndrome, and psoriatic arthritis),autoimmune gastrointestinal and liver disorders (such as, for example,inflammatory bowel diseases (e.g., ulcerative colitis and Crohn'sdisease), autoimmune gastritis and pernicious anemia, autoimmunehepatitis, primary biliary cirrhosis, primary sclerosing cholangitis,and celiac disease), vasculitis (such as, for example, ANCA-negativevasculitis and ANCA-associated vasculitis, including Churg-Straussvasculitis, Wegener's granulomatosis, and microscopic polyangiitis),autoimmune neurological disorders (such as, for example, multiplesclerosis, opsoclonus myoclonus syndrome, myasthenia gravis,neuromyelitis optica, Parkinson's disease, Alzheimer's disease, andautoimmune polyneuropathies), renal disorders (such as, for example,glomerulonephritis, Goodpasture's syndrome, and Berger's disease),autoimmune dermatologic disorders (such as, for example, psoriasis,urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneouslupus erythematosus), hematologic disorders (such as, for example,thrombocytopenic purpura, thrombotic thrombocytopenic purpura,post-transfusion purpura, and autoimmune hemolytic anemia),atherosclerosis, uveitis, autoimmune hearing diseases (such as, forexample, inner ear disease and hearing loss), Behcet's disease,Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders(such as, for example, diabetic-related autoimmune diseases such asinsulin-dependent diabetes mellitus (IDDM), Addison's disease, andautoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, rheumatoid arthritis,ulcerative colitis, ANCA-associated vasculitis, lupus, multiplesclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

Specific examples of other autoimmune diseases as defined herein, whichin some cases encompass those listed above, include, but are not limitedto, arthritis (acute and chronic, rheumatoid arthritis includingjuvenile-onset rheumatoid arthritis and stages such as rheumatoidsynovitis, gout or gouty arthritis, acute immunological arthritis,chronic inflammatory arthritis, degenerative arthritis, type IIcollagen-induced arthritis, infectious arthritis, Lyme arthritis,proliferative arthritis, psoriatic arthritis, Still's disease, vertebralarthritis, osteoarthritis, arthritis chronica progrediente, arthritisdeformans, polyarthritis chronica primaria, reactive arthritis,menopausal arthritis, estrogen-depletion arthritis, and ankylosingspondylitis/rheumatoid spondylitis), autoimmune lymphoproliferativedisease, inflammatory hyperproliferative skin diseases, psoriasis suchas plaque psoriasis, gutatte psoriasis, pustular psoriasis, andpsoriasis of the nails, atopy including atopic diseases such as hayfever and Job's syndrome, dermatitis including contact dermatitis,chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis,allergic contact dermatitis, hives, dermatitis herpetiformis, nummulardermatitis, seborrheic dermatitis, non-specific dermatitis, primaryirritant contact dermatitis, and atopic dermatitis, x-linked hyper IgMsyndrome, allergic intraocular inflammatory diseases, urticaria such aschronic allergic urticaria and chronic idiopathic urticaria, includingchronic autoimmune urticaria, myositis, polymyositis/dermatomyositis,juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma(including systemic scleroderma), sclerosis such as systemic sclerosis,multiple sclerosis (MS) such as spino-optical MS, primary progressive MS(PPMS), and relapsing remitting MS (RRMS), progressive systemicsclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata,ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease(IBD) (for example, Crohn's disease, autoimmune-mediatedgastrointestinal diseases, gastrointestinal inflammation, colitis suchas ulcerative colitis, colitis ulcerosa, microscopic colitis,collagenous colitis, colitis polyposa, necrotizing enterocolitis, andtransmural colitis, and autoimmune inflammatory bowel disease), bowelinflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosingcholangitis, respiratory distress syndrome, including adult or acuterespiratory distress syndrome (ARDS), meningitis, inflammation of all orpart of the uvea, iritis, choroiditis, an autoimmune hematologicaldisorder, graft-versus-host disease, angioedema such as hereditaryangioedema, cranial nerve damage as in meningitis, herpes gestationis,pemphigoid gestationis, pruritis scroti, autoimmune premature ovarianfailure, sudden hearing loss due to an autoimmune condition,IgE-mediated diseases such as anaphylaxis and allergic and atopicrhinitis, encephalitis such as Rasmussen's encephalitis and limbicand/or brainstem encephalitis, uveitis, such as anterior uveitis, acuteanterior uveitis, granulomatous uveitis, nongranulomatous uveitis,phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis such as primary GN, immune-mediatedGN, membranous GN (membranous nephropathy), idiopathic membranous GN oridiopathic membranous nephropathy, membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressiveGN(RPGN), proliferative nephritis, autoimmune polyglandular endocrinefailure, balanitis including balanitis circumscripta plasmacellularis,balanoposthitis, erythema annulare centrifugum, erythema dyschromicumperstans, eythema multiform, granuloma annulare, lichen nitidus, lichensclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus,lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis,premalignant keratosis, pyoderma gangrenosum, allergic conditions andresponses, food allergies, drug allergies, insect allergies, rareallergic disorders such as mastocytosis, allergic reaction, eczemaincluding allergic or atopic eczema, asteatotic eczema, dyshidroticeczema, and vesicular palmoplantar eczema, asthma such as asthmabronchiale, bronchial asthma, and auto-immune asthma, conditionsinvolving infiltration of T cells and chronic inflammatory responses,immune reactions against foreign antigens such as fetal A-B-O bloodgroups during pregnancy, chronic pulmonary inflammatory disease,autoimmune myocarditis, leukocyte adhesion deficiency, lupus, includinglupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus,extra-renal lupus, discoid lupus and discoid lupus erythematosus,alopecia lupus, SLE, such as cutaneous SLE or subacute cutaneous SLE,neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus,juvenile onset (Type I) diabetes mellitus, including pediatric IDDM,adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes,idiopathic diabetes insipidus, diabetic retinopathy, diabeticnephropathy, diabetic colitis, diabetic large-artery disorder, immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosisincluding lymphomatoid granulomatosis, agranulocytosis, vasculitides(including large-vessel vasculitis such as polymyalgia rheumatica andgiant-cell (Takayasu's) arteritis, medium-vessel vasculitis such asKawasaki's disease and polyarteritis nodosa/periarteritis nodosa,immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivityvasculitis, necrotizing vasculitis such as fibrinoid necrotizingvasculitis and systemic necrotizing vasculitis, ANCA-negativevasculitis, and ANCA-associated vasculitis such as Churg-Strausssyndrome (CSS), Wegener's granulomatosis, and microscopic polyangiitis),temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombspositive anemia, Diamond Blackfan anemia, hemolytic anemia or immunehemolytic anemia including autoimmune hemolytic anemia (AIHA),pernicious anemia (anemia perniciosa), Addison's disease, pure red cellanemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A,autoimmune neutropenia(s), cytopenias such as pancytopenia, leukopenia,diseases involving leukocyte diapedesis, CNS inflammatory disorders,Alzheimer's disease, Parkinson's disease, multiple organ injury syndromesuch as those secondary to septicemia, trauma or hemorrhage,antigen-antibody complex-mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, motoneuritis,allergic neuritis, Behcet's disease/syndrome, Castleman's syndrome,Goodpasture's syndrome, Reynaud's syndrome, Sjögren's syndrome,Stevens-Johnson syndrome, pemphigoid or pemphigus such as pemphigoidbullous, cicatricial (mucous membrane) pemphigoid, skin pemphigoid,pemphigus vulgaris, paraneoplastic pemphigus, pemphigus foliaceus,pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus,epidermolysis bullosa acquisita, ocular inflammation, preferablyallergic ocular inflammation such as allergic conjunctivis, linear IgAbullous disease, autoimmune-induced conjunctival inflammation,autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermalinjury due to an autoimmune condition, preeclampsia, an immune complexdisorder such as immune complex nephritis, antibody-mediated nephritis,neuroinflammatory disorders, polyneuropathies, chronic neuropathy suchas IgM polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (asdeveloped by myocardial infarction patients, for example), includingthrombotic thrombocytopenic purpura (TTP), post-transfusion purpura(PTP), heparin-induced thrombocytopenia, and autoimmune orimmune-mediated thrombocytopenia including, for example, idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP, scleritissuch as idiopathic cerato-scleritis, episcleritis, autoimmune disease ofthe testis and ovary including autoimmune orchitis and oophoritis,primary hypothyroidism, hypoparathyroidism, autoimmune endocrinediseases including thyroiditis such as autoimmune thyroiditis,Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), orsubacute thyroiditis, autoimmune thyroid disease, idiopathichypothyroidism, Grave's disease, Grave's eye disease (ophthalmopathy orthyroid-associated ophthalmopathy), polyglandular syndromes such asautoimmune polyglandular syndromes, for example, type I (orpolyglandular endocrinopathy syndromes), paraneoplastic syndromes,including neurologic paraneoplastic syndromes such as Lambert-Eatonmyasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-personsyndrome, encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, pneumonitis such as lymphoidinterstitial pneumonitis (LIP), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barré syndrome, Berger's disease (IgAnephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acutefebrile neutrophilic dermatosis, subcorneal pustular dermatosis,transient acantholytic dermatosis, cirrhosis such as primary biliarycirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiacor Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia such as mixed cryoglobulinemia,amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronaryartery disease, autoimmune ear disease such as autoimmune inner eardisease (AIED), autoimmune hearing loss, polychondritis such asrefractory or relapsed or relapsing polychondritis, pulmonary alveolarproteinosis, keratitis such as Cogan's syndrome/nonsyphiliticinterstitial keratitis, Bell's palsy, Sweet's disease/syndrome, rosaceaautoimmune, zoster-associated pain, amyloidosis, a non-cancerouslymphocytosis, a primary lymphocytosis, which includes monoclonal B celllymphocytosis (e.g., benign monoclonal gammopathy and monoclonalgammopathy of undetermined significance, MGUS), peripheral neuropathy,paraneoplastic syndrome, channelopathies such as epilepsy, migraine,arrhythmia, muscular disorders, deafness, blindness, periodic paralysis,and channelopathies of the CNS, autism, inflammatory myopathy, focal orsegmental or focal segmental glomerulosclerosis (FSGS), endocrineophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatologicaldisorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome,adrenalitis, gastric atrophy, presenile dementia, demyelinating diseasessuch as autoimmune demyelinating diseases and chronic inflammatorydemyelinating polyneuropathy, Dressler's syndrome, alopecia greata,alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon,esophageal dysmotility, sclerodactyl), and telangiectasia), male andfemale autoimmune infertility, e.g., due to anti-spermatozoanantibodies, mixed connective tissue disease, Chagas' disease, rheumaticfever, recurrent abortion, farmer's lung, erythema multiforme,post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung,allergic granulomatous angiitis, benign lymphocytic angiitis, Alport'ssyndrome, alveolitis such as allergic alveolitis and fibrosingalveolitis, interstitial lung disease, transfusion reaction, leprosy,malaria, parasitic diseases such as leishmaniasis, kypanosomiasis,schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan'ssyndrome, dengue, endocarditis, endomyocardial fibrosis, diffuseinterstitial pulmonary fibrosis, interstitial lung fibrosis, fibrosingmediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cysticfibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, flariasis, cyclitis such as chronic cyclitis,heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch'scyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV)infection, SCID, acquired immune deficiency syndrome (AIDS), echovirusinfection, sepsis (systemic inflammatory response syndrome (SIRS)),endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubellavirus infection, post-vaccination syndromes, congenital rubellainfection, Epstein-Barr virus infection, mumps, Evan's syndrome,autoimmune gonadal failure, Sydenham's chorea, post-streptococcalnephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis,chorioiditis, giant-cell polymyalgia, chronic hypersensitivitypneumonitis, conjunctivitis, such as vernal catarrh,keratoconjunctivitis sicca, and epidemic keratoconjunctivitis,idiopathic nephritic syndrome, minimal change nephropathy, benignfamilial and ischemia-reperfusion injury, transplant organ reperfusion,retinal autoimmunity, joint inflammation, bronchitis, chronicobstructive airway/pulmonary disease, silicosis, aphthae, aphthousstomatitis, arteriosclerotic disorders (cerebral vascular insufficiency)such as arteriosclerotic encephalopathy and arterioscleroticretinopathy, aspermiogenese, autoimmune hemolysis, Boeck's disease,cryoglobulinemia, Dupuytren's contracture, endophthalmiaphacoanaphylactica, enteritis allergica, erythema nodosum leprosum,idiopathic facial paralysis, chronic fatigue syndrome, febrisrheumatica, Hamman-Rich's disease, sensoneural hearing loss,haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica (sympatheticophthalmitis), neonatal ophthalmitis, optic neuritis, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,non-malignant thymoma, lymphofollicular thymitis, vitiligo, toxic-shocksyndrome, food poisoning, conditions involving infiltration of T cells,leukocyte-adhesion deficiency, immune responses associated with acuteand delayed hypersensitivity mediated by cytokines and T-lymphocytes,diseases involving leukocyte diapedesis, multiple organ injury syndrome,antigen-antibody complex-mediated diseases, antiglomerular basementmembrane disease, autoimmune polyendocrinopathies, oophoritis, primarymyxedema, autoimmune atrophic gastritis, rheumatic diseases, mixedconnective tissue disease, nephrotic syndrome, insulitis, polyendocrinefailure, autoimmune polyglandular syndromes, including polyglandularsyndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH),cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosaacquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome,primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acuteor chronic sinusitis, ethmoid, frontal, maxillary, or sphenoidsinusitis, allergic sinusitis, an eosinophil-related disorder such aseosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgiasyndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropicalpulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, orgranulomas containing eosinophils, anaphylaxis, spondyloarthropathies,seronegative spondyloarthritides, polyendocrine autoimmune disease,sclerosing cholangitis, sclera, episclera, chronic mucocutaneouscandidiasis, Bruton's syndrome, transient hypogammaglobulinemia ofinfancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome,angiectasis, autoimmune disorders associated with collagen disease,rheumatism such as chronic arthrorheumatism, lymphadenitis, reduction inblood pressure response, vascular dysfunction, tissue injury,cardiovascular ischemia, hyperalgesia, renal ischemia, cerebralischemia, and disease accompanying vascularization, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,ischemic re-perfusion disorder, reperfusion injury of myocardial orother tissues, lymphomatous tracheobronchitis, inflammatory dermatoses,dermatoses with acute inflammatory components, multiple organ failure,bullous diseases, renal cortical necrosis, acute purulent meningitis orother central nervous system inflammatory disorders, ocular and orbitalinflammatory disorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, narcolepsy, acute serious inflammation,chronic intractable inflammation, pyelitis, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis. Such diseases arecontemplated herein to be treated by the administration of an antibodywhich binds to a B cell surface marker, such as CD22, and includes theadministration of an unconjugated (“naked”) antibody or an antibodyconjugated to a cytotoxic agent as disclosed herein. Such diseases arealso contemplated herein to be treated by combination therapy includingan anti-CD22 antibody or anti-CD22 antibody drug conjugate of theinvention in combination with another antibody or antibody drugconjugate, another cytoxic agent, radiation or other treatmentadministered simultaneously or in series.

As used herein, “treatment” (and variations such as “treat” or“treating”) refers to clinical intervention in an attempt to alter thenatural course of the individual or cell being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies of the invention are used to delaydevelopment of a disease or disorder or to slow the progression of adisease or disorder.

An “individual” is a vertebrate. In certain embodiments, the vertebrateis a mammal. Mammals include, but are not limited to, farm animals (suchas cows), sport animals, pets (such as cats, dogs, and horses),primates, mice and rats. In certain embodiments, a mammal is a human.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thesubstance/molecule, to elicit a desired response in the individual. Atherapeutically effective amount encompasses an amount in which anytoxic or detrimental effects of the substance/molecule are outweighed bythe therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,but not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount would be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu), chemotherapeutic agents (e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents, enzymes and fragmentsthereof such as nucleolytic enzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including fragments and/or variantsthereof, toxins, growth inhibitory agents, drug moieties, and thevarious antitumor or anticancer agents disclosed below. Other cytotoxicagents are described below. A tumoricidal agent causes destruction oftumor cells.

A “toxin” is any substance capable of having a detrimental effect on thegrowth or proliferation of a cell.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTINO), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosoureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and 5(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens suchas calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene,Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine(ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol;mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa;taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology,Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineerednanoparticle formulation of paclitaxel (American PharmaceuticalPartners, Schaumberg, Ill.), and TAXOTERE® docetaxel (Rhône-PoulencRorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®);6-thioguanine; mercaptopurine; methotrexate; platinum analogs such ascisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin;leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate;daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid;capecitabine (XELODA®); pharmaceutically acceptable salts, acids orderivatives of any of the above; as well as combinations of two or moreof the above such as CHOP, an abbreviation for a combined therapy ofcyclophosphamide, doxorubicin, vincristine, and prednisolone, andFOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell (such as a cell expressingCD22) either in vitro or in vivo. Thus, the growth inhibitory agent maybe one which significantly reduces the percentage of cells (such as acell expressing CD22) in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

The term “intracellular metabolite” refers to a compound resulting froma metabolic process or reaction inside a cell on an antibody-drugconjugate (ADC). The metabolic process or reaction may be an enzymaticprocess, such as proteolytic cleavage of a peptide linker of the ADC, orhydrolysis of a functional group such as a hydrazone, ester, or amide.Intracellular metabolites include, but are not limited to, antibodiesand free drug which have undergone intracellular cleavage after entry,diffusion, uptake or transport into a cell.

The terms “intracellularly cleaved” and “intracellular cleavage” referto a metabolic process or reaction inside a cell on an antibody-drugconjugate (ADC) whereby the covalent attachment, i.e. linker, betweenthe drug moiety (D) and the antibody (Ab) is broken, resulting in thefree drug dissociated from the antibody inside the cell. The cleavedmoieties of the ADC are thus intracellular metabolites.

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

The term “cytotoxic activity” refers to a cell-killing, cytostatic orgrowth inhibitory effect of an antibody-drug conjugate or anintracellular metabolite of an antibody-drug conjugate. Cytotoxicactivity may be expressed as the IC₅₀ value, which is the concentration(molar or mass) per unit volume at which half the cells survive.

“Alkyl” is C1-C18 hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH3), ethyl (Et,—CH2CH3), 1-propyl (n-Pr, n-propyl, —CH2CH2CH3), 2-propyl (i-Pr,i-propyl, —CH(CH3)2), 1-butyl (n-Bu, n-butyl, —CH2CH2CH2CH3),2-methyl-1-propyl (i-Bu, i-butyl, —CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl,—CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH3)3), 1-pentyl(n-pentyl, —CH2CH2CH2CH2CH3), 2-pentyl (—CH(CH3)CH2CH2CH3), 3-pentyl(—CH(CH2CH3)2), 2-methyl-2-butyl (—C(CH3)2CH2CH3), 3-methyl-2-butyl(—CH(CH3)CH(CH3)2), 3-methyl-1-butyl (—CH2CH2CH(CH3)2), 2-methyl-1-butyl(—CH2CH(CH3)CH2CH3), 1-hexyl (—CH2CH2CH2CH2CH2CH3), 2-hexyl(—CH(CH3)CH2CH2CH2CH3), 3-hexyl (—CH(CH2CH3)(CH2CH2CH3)),2-methyl-2-pentyl (—C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl(—CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (—CH(CH3)CH2CH(CH3)2),3-methyl-3-pentyl (—C(CH3)(CH2CH3)2), 2-methyl-3-pentyl(—CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (—C(CH3)2CH(CH3)2),3,3-dimethyl-2-butyl (—CH(CH3)C(CH3)3.

The term “C₁-C₈ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 8 carbonatoms. Representative “C₁-C₈ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl,-n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C₁-C₈ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C₁-C₈ alkylsinclude, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl,-isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl,-2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl, -3-methyl-1 butynyl. methyl, ethyl, propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl,3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl,3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl,n-heptyl, isoheptyl, n-octyl, and isooctyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where eachR′ is independently selected from H, —C₁-C₈ alkyl and aryl.

“Alkenyl” is C2-C18 hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H₇), and 5-hexenyl (—CH₂ CH₂CH₂CH₂CH═CH₂)

“Alkynyl” is C2-C18 hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to:acetylenic (—CCH) and propargyl (—CH₂C≡CH),

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—) 1,2-ethyl,(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbon group ofthe formula —(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡C—).

“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracenyl. Acarbocyclic aromatic group or a heterocyclic aromatic group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R)₂ and —CN; wherein each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkylmoiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkylgroup is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbonatoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety ofthe heteroarylalkyl group may be a monocycle having 3 to 7 ring members(2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), forexample: a bicyclo[4,5], [5,5], [5,6], or [6,6] system.

“Substituted alkyl,” “substituted aryl,” and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a substituent.Typical substituents include, but are not limited to, —X, —R, —O⁻, —OR,—SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO,—NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R, —C(═O)NR₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R,—OS(═O)₂OR, —S(═O)₂NR, —S(═O)R, —OP(═O)(OR)₂, —P(═O)(OR)₂, —PO⁻ ₃,—PO₃H₂, —C(═O)R, —C(═O)X, —C(═S)R, —CO₂R, —CO₂ ⁻, —C(═S)OR, —C(═O)SR,—C(═S)SR, —C(═O)NR₂, —C(═S)NR₂, —C(═NR)NR₂, where each X isindependently a halogen: F, Cl, Br, or I; and each R is independently—H, C₂-C₁₈ alkyl, C₆-C₂₀ aryl, C₃-C₁₄ heterocycle, protecting group orprodrug moiety. Alkylene, alkenylene, and alkynylene groups as describedabove may also be similarly substituted.

“Heteroaryl” and “heterocycle” refer to a ring system in which one ormore ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 1 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo[4,5], [5,5], [5,6], or [6,6]system.

Heterocycles are described in Paquette, Leo A.; “Principles of ModernHeterocyclic Chemistry” (W.A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.(1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₈ heterocyclo” refers to a C₃-C₈ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up tosix groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbonatoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocycliccarbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ringatoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g. arranged as abicyclo[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo[5,6] or [6,6] system. Examples of monocycliccarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated orunsaturated non-aromatic carbocyclic ring. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,-C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined abovewherein one of the carbocycle groups' hydrogen atoms is replaced with abond.

“Linker” refers to a chemical moiety comprising a covalent bond or achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, linkers include a divalent radical such as analkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:—(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g. polyethylenoxy,PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino,Jeffamine™); and diacid ester and amides including succinate,succinamide, diglycolate, malonate, and caproamide.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and l or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or l meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Certain leaving groups are well known in theart, and examples include, but are not limited to, a halide (e.g.,chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

ABBREVIATIONS

Linker Components:

-   -   MC=6-maleimidocaproyl    -   Val-Cit or “vc”=valine-citrulline (an exemplary dipeptide in a        protease cleavable linker)    -   Citrulline=2-amino-5-ureido pentanoic acid    -   PAB=p-aminobenzyloxycarbonyl (an example of a “self immolative”        linker component)    -   Me-Val-Cit ═N-methyl-valine-citrulline (wherein the linker        peptide bond has been modified to prevent its cleavage by        cathepsin B)    -   MC(PEG)6-OH=maleimidocaproyl-polyethylene glycol (can be        attached to antibody cysteines).    -   SPP=N-succinimidyl-4-(2-pyridylthio)pentanoate    -   SPDP=N-succinimidyl-3-(2-pyridyldithio) propionate    -   SMCC=succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate    -   IT=iminothiolane

Cytotoxic Drugs:

-   -   MMAE=mono-methyl auristatin E (MW 718)    -   MMAF=variant of auristatin E (MMAE) with a phenylalanine at the        C-terminus of the drug (MW 731.5)    -   MMAF-DMAEA=MMAF with DMAEA (dimethylaminoethylamine) in an amide        linkage to the C-terminal phenylalanine (MW 801.5)    -   MMAF-TEG=MMAF with tetraethylene glycol esterified to the        phenylalanine    -   MMAF-NtBu=N-t-butyl, attached as an amide to C-terminus of MMAF    -   DM1=N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine    -   DM3=N(2)-deacetyl-N2-(4-mercapto-1-oxopentyl)-maytansine

DM4=N(2)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine

Further abbreviations are as follows: AE is auristatin E, Boc isN-(t-butoxycarbonyl), cit is citrulline, dap is dolaproine, DCC is1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA isdiethylamine, DEAD is diethylazodicarboxylate, DEPC isdiethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA isN,N-diisopropylethylamine, dil is dolaisoleucine, DMA isdimethylacetamide, DMAP is 4-dimethylaminopyridine, DME isethyleneglycol dimethyl ether (or 1,2-dimethoxyethane), DMF isN,N-dimethylformamide, DMSO is dimethylsulfoxide, doe is dolaphenine,dov is N,N-dimethylvaline, DTNB is 5,5′-dithiobis(2-nitrobenzoic acid),DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCIis 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is2-ethoxy-1-ethyoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospraymass spectrometry, EtOAc is ethyl acetate, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-,N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HOBt is 1hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine,MeCN(CH₃CN) is acetonitrile, MeOH is methanol, Mtr is4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is(1S,2R)-(+)-norephedrine, PBS is phosphate-buffered saline (pH 7.4), PEGis polyethylene glycol, Ph is phenyl, Pnp is p-nitrophenyl, MC is6-maleimidocaproyl, phe is L-phenylalanine, PyBrop is bromotris-pyrrolidino phosphonium hexafluorophosphate, SEC is size-exclusionchromatography, Su is succinimide, TFA is trifluoroacetic acid, TLC isthin layer chromatography, UV is ultraviolet, and val is valine.

Compositions and Methods of Making the Same

Antibodies that bind to CD22 are provided. Immunoconjugates comprisinganti-CD22 antibodies are provided. Antibodies and immunoconjugates ofthe invention are useful, e.g., for the diagnosis or treatment ofdisorders associated with altered expression, e.g., increasedexpression, of CD22. In certain embodiments, antibodies orimmunoconjugates of the invention are useful for the diagnosis ortreatment of a cell proliferative disorder, such as cancer.

Anti-CD22 Antibodies

In one aspect, the invention provides antibodies that bind to CD22. Insome embodiments, antibodies are provided that bind to a mature form ofhuman and cynomolgus monkey (cyno) CD22. In one such embodiment, amature form of human CD22 has an amino acid sequence of SEQ ID NO:27.The mature, major human isoform has an extracellular domain comprisingseven Ig-like domains and an amino acid sequence of SEQ ID NO:28. Inanother embodiment, a minor isoform of human CD22 lacking extracellulardomains 3 and 4 has an amino acid sequence of SEQ ID NO: 29. The aminoacid sequence of the extracellular domain of the minor isoform is SEQ IDNO:30. The cyno CD22 has an amino acid sequence of SEQ ID NO:31. In someembodiments, an antibody to CD22 binds to a mature form of CD22expressed on the cell surface. In some embodiments, an antibody thatbinds to a mature form of CD22 expressed on the cell surface inhibitsthe growth of the cell. In some embodiments, an anti-CD22 antibody bindsto a mature form of CD22 expressed on the cell surface and inhibits cellproliferation. In certain embodiments, an anti-CD22 antibody binds to amature form of CD22 expressed on the cell surface and induces celldeath. In some embodiments, an anti-CD22 antibody binds to a mature formof CD22 expressed on the surface of cancer cells. In some embodiments,an anti-CD22 antibody binds to a mature form of CD22 that isoverexpressed on the surface of cancer cells relative to normal cells ofthe same tissue origin. In some embodiments, an anti-CD22 antibody isconjugated to a cytotoxin or a detectable label and binds to CD22 on acell surface. In some embodiments, the antibody-toxin conjugate inhibitsgrowth of the cell. In some embodiments, the antibody-detectable labelconjugate causes a cell expressing CD22 on its surface to be detectablein vitro or in vivo.

In one aspect, an anti-CD22 antibody is a monoclonal antibody. In oneaspect, an anti-CD22 antibody is an antibody fragment, e.g., a Fab,Fab′-SH, Fv, scFv, or (Fab′)₂ fragment. In one aspect, an anti-CD22antibody is a chimeric, humanized, or human antibody. In one aspect, anyof the anti-CD22 antibodies described herein are purified.

Exemplary monoclonal antibodies derived from a phage library areprovided herein. The antigen used for screening the library was apolypeptide having the sequence of amino acid sequences of SEQ ID NO:28or SEQ ID NO:30, corresponding to the extracellular domains (ECDs) ofCD22 beta and alpha. The antibodies resulting from the library screenare affinity matured.

In one aspect, monoclonal antibodies that compete with murine 10F4.4.1,humanized 10F4v1 and v3, and murine 5E8.1.8 for binding to CD22 areprovided. Monoclonal antibodies that bind to the same epitope as murine10F4.4.1, humanized 10F4v1 and v3, and murine 5E8.1.8 are also provided.

In one aspect of the invention, polynucleotides encoding anti-CD22antibodies are provided. In certain embodiments, vectors comprisingpolynucleotides encoding anti-CD22 antibodies are provided. In certainembodiments, host cells comprising such vectors are provided. In anotheraspect of the invention, compositions comprising anti-CD22 antibodies orpolynucleotides encoding anti-CD22 antibodies are provided. In certainembodiments, a composition of the invention is a pharmaceuticalformulation for the treatment of a cell proliferative disorder, such asthose enumerated herein.

Antibody Administration and Formulation

In one embodiment, the anti-CD22 antibody or anti-CD22 antibody drugconjugate (including, but not limited to, an anti-CD22 thiomab drugconjugate of the invention) of the invention is administered incombination with an antagonist of a B-cell surface antigen.Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order. In one embodiment, the administration is consecutive orsequential. In another embodiment, the administration is simultaneous,concurrent, or together in the same formulation. In one embodiment, theB-cell surface antigen antagonist is an antibody or antigen bindingfragment thereof. In one embodiment, the B-cell surface antagonist is anantibody drug conjugate.

The formulations herein may contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, in addition to an anti-CD22 antibody, anti-CD22 antibodydrug conjugate or CD22 binding oligopeptide, it may be desirable toinclude in the one formulation, an additional antibody, e.g., a secondanti-CD22 antibody which binds a different epitope on the CD22polypeptide, or a second antibody that binds a different B-cell surfaceantigen, or an antibody to some other target such as a growth factorthat affects the growth of the particular cancer. Alternatively, oradditionally, the composition may further comprise a chemotherapeuticagent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonalagent, and/or cardioprotectant. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

Currently, depending on the stage of the cancer, cancer treatmentinvolves one or a combination of the following therapies: surgery toremove the cancerous tissue, radiation therapy, and chemotherapy.Anti-CD22 antibody, anti-CD22 antibody drug conjugate or oligopeptidetherapy may be especially desirable in elderly patients who do nottolerate the toxicity and side effects of chemotherapy well and inmetastatic disease where radiation therapy has limited usefulness. Thetumor targeting anti-CD22 antibodies, anti-CD22 antibody drug conjugateor oligopeptide of the invention are useful to alleviate CD22 cancersupon initial diagnosis of the disease or during relapse. For therapeuticapplications, the anti-CD22 antibody, anti-CD22 antibody drug conjugateor oligopeptide can be used alone, or in combination therapy with, e.g.,hormones, antiangiogens, or radiolabelled compounds, or with surgery,cryotherapy, and/or radiotherapy. Anti-CD22 antibody, anti-CD22 antibodydrug conjugate or oligopeptide treatment can be administered inconjunction with other forms of conventional therapy, eitherconsecutively with, pre- or post-conventional therapy. In the presentmethod of the invention for treating or alleviating cancer, the cancerpatient can be administered anti-CD22 antibody, anti-CD22 antibody drugconjugate or oligopeptide in conjuction with treatment with the one ormore of the preceding chemotherapeutic agents. The anti-CD22 antibody,anti-CD22 antibody drug conjugate or oligopeptide will be administeredwith a therapeutically effective dose of the chemotherapeutic agent. Inanother embodiment, the anti-CD22 antibody, anti-CD22 antibody drugconjugate or oligopeptide is administered in conjunction withchemotherapy to enhance the activity and efficacy of thechemotherapeutic agent. The Physicians' Desk Reference (PDR) disclosesdosages of these agents that have been used in treatment of variouscancers. The dosing regimen and dosages of these aforementionedchemotherapeutic drugs that are therapeutically effective will depend onthe particular cancer being treated, the extent of the disease and otherfactors familiar to the physician of skill in the art and can bedetermined by the physician.

In one particular embodiment, a conjugate comprising an anti-CD22antibody, anti-CD22 antibody drug conjugate or oligopeptide conjugatedwith a cytotoxic agent is administered to the patient. Preferably, theimmunoconjugate bound to the CD22 protein is internalized by the cell,resulting in increased therapeutic efficacy of the immunoconjugate inkilling the cancer cell to which it binds. In one embodiment, thecytotoxic agent targets or interferes with the nucleic acid in thecancer cell. Examples of cytotoxic agents are described above andinclude auristatins, maytansinoids, calicheamicins, ribonucleases andDNA endonucleases, or biologically active derivatives thereof.

The anti-CD22 antibodies, anti-CD22 antibody drug conjugates oroligopeptides or toxin conjugates thereof are administered to a humanpatient, in accord with known methods, such as intravenousadministration, e.g.,, as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody, anti-CD22 antibody drug conjugate oroligopeptide is preferred.

Other therapeutic regimens may be combined with the administration ofthe anti-CD22 antibody, anti-CD22 antibody drug conjugate oroligopeptide. The combined administration includes co-administration,using separate formulations or a single pharmaceutical formulation, andconsecutive administration in either order, wherein preferably there isa time period while both (or all) active agents simultaneously exerttheir biological activities. Preferably such combined therapy results ina synergistic therapeutic effect.

It may also be desirable to combine administration of the anti-CD22antibody or antibodies, anti-CD22 antibody drug conjugates oroligopeptides, with administration of an antibody directed againstanother tumor antigen or B-cell surface antigen associated with theparticular cancer.

In another embodiment, the therapeutic treatment methods of the presentinvention involves the combined administration of an anti-CD22 antibody(or antibodies), anti-CD22 antibody drug conjugate(s) or oligopeptide(s)and one or more chemotherapeutic agents or growth inhibitory agents,including co-administration of cocktails of different chemotherapeuticagents. Chemotherapeutic agents include estramustine phosphate,prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide,hydroxyurea and hydroxyureataxanes (such as paclitaxel and doxetaxel)and/or anthracycline antibiotics, as well as combinations of agents suchas, but not limited to, CHOP or FOLFOX. Preparation and dosing schedulesfor such chemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,Baltimore, Md. (1992).

The antibody is administered by any suitable means, includingparenteral, topical, subcutaneous, intraperitoneal, intrapulmonary,intranasal, and/or intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Intrathecal administration is alsocontemplated (see, e.g., US Patent Appln No. 2002/0009444, Grillo-Lopez,A, concerning intrathecal delivery of a CD20 antibody). Preferably, thedosing is given intravenously or subcutaneously.

A second medicament may be administered with the initial exposure and/orlater exposures of the therapeutic antibody or immunoadhesin, suchcombined administration includes co-administration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities.

While the therapeutic anti-CD22 antibody, anti-CD22 antibody drugconjugate, immunoadhesin or other biologic may be administered as asingle-agent to treat the autoimmune disease, generally, the therapeuticantibody or immunoadhesin will be combined with one or more secondmedicament(s). For example, for RA, and other autoimmune diseases, theantibody, immunoadhesin, or other biologic drug is preferably combinedwith any one or more of the immunosuppressive agents, chemotherapeuticagents, BAFF antagonists, integrin antagonists or antibodies, and/orcytokines listed in the definitions section above; any one or moredisease-modifying antirheumatic drugs (DMARDs), such ashydroxycloroquine, sulfasalazine, methotrexate, leflunomide,azathioprine, D-penicillamine, Gold (oral), Gold (intramuscular),minocycline, cyclosporine; Staphylococcal protein A immunoadsorption;intravenous immunoglobulin (IVIG); nonsteroidal antiinflammatory drugs(NSAIDs); glucocorticoid (e.g. via joint injection); corticosteroid(e.g. methylprednisolone and/or prednisone); folate; an anti-tumornecrosis factor (TNF) antagonist, e.g. etanercept/ENBREL™,infliximab/REMICADE™, D2E7 (Knoll) or CDP-870 (Celltech); IL-1Rantagonist (e.g. Kineret); IL-10 antagonist (e.g. Ilodecakin); a bloodclotting modulator (e.g. WinRho); an IL-6 antagonist/anti-TNF (CBP1011); CD40 antagonist (e.g. IDEC 131); Ig-Fc receptor antagonist(MDX33); immunomodulator (e.g. thalidomide or ImmuDyn); anti-CD5antibody (e.g. H5g1.1); macrophage inhibitor (e.g. MDX 33);costimulatory blocker (e.g. BMS 188667 or Tolerimab); complementinhibitor (e.g. h5G1.1, 3E10 or an anti-decay accelerating factor (DAF)antibody); IL-2 antagonist (zxSMART); EGFR inhibitor (see definitionabove); tyrosine kinase inhibitor (see definition above);anti-angiogenic agent (e.g. VEGF antibody such as bevacizumab); CD22antibodies such as LL2 or epratuzumab (LYMPHOCIDE®; Immunomedics),including epratuzumab Y-90 (Juweid et al. Cancer Res 55 (23Suppl):5899s-5907s (1995)), Abiogen's CD22 antibody (Abiogen, Italy),CMC 544 (Wyeth/Celltech), combotox (UT Soutwestern), BL22 (NIH), andLympoScan Tc99 (Immunomedics); EpCAM antibody such as 17-1A (PANOREX®);αcβ3 antibody (e.g. VITAXIN®; Medimmune); CD37 antibody such as TRU 016(Trubion); IL-21 antibody (Zymogenetics/Novo Nordisk); anti-B cellantibody (Impheron); B cell targeting MAb (Immunogen/Aventis); 1D09C3(Morphosys/GPC); LymphoRad 131 (HGS); Lym-1 antibody Y-90 (USC); LIF 226(Enhanced Lifesci.); BAFF antibody (e.g., WO 03/33658); BAFF receptorantibody (e.g., WO 02/24909); BR3 antibody; Blys antibody such asbelimumab; LYMPHOSCD22-B™; anti-Lym-1 Oncolym (USC/Peregrine); ISF 154(UCSD/Roche/Tragen); gomilixima (Idec 152; Biogen Idec); IL-6 receptorantibody such as atlizumab (ACTEMRA™; Chugai/Roche); IL-15 antibody suchas HuMax-Il-15 (Genmab/Amgen); chemokine receptor antibody, such as aCCR2 antibody (e.g. MLN1202; Millieneum); anti-complement antibody, suchas C5 antibody (e.g. eculizumab, 5G1.1; Alexion); oral formulation ofhuman immunoglobulin (e.g. IgPO; Protein Therapeutics); IL-12 antibodysuch as ABT-874 (CAT/Abbott); Teneliximab (BMS-224818); B cell vaccine;DN-BAFF (Xencor); CRx-119 (CombinatoRx); Amgen's BAFF antagonist;Pentostatin (Pfizer); IC-485 (ICOS); chemokine antagonist such as T-487(Tularik) or Reticulose (AVR-118); SCO-323 (SCIOS); integrin antagonist683699, Tanabe, NGD-2001-1 (Neurogen); SCID-469 (SCIOS); BIRB-796(Boehringer Ingelheim); VX702, VX850 (Vertex); Leukotriene B-4antagonist (such as amelubunt, BIM-284; BI); microtubule modulator(Paxceed; Angiotech); protease inhibitor (MBS561392; BMS); AGIX-4207(Atherogenics); ISIS-104838 (ISIS/Elan); MFG-IRAP (Univ. Pitt.); IL-1Trap (RGN-303; Regeneron/Novartis); oprelvekin (Wyeth); everolimus(Certican; Novartis); Amevive (Biogen Idec); ORG-39141 (Organon); FK-506(Fujisawa); and IL-2 antagonist (tacrolimus; Fujisawa).

A detailed description of exemplary anti-CD22 antibodies is as follows:

1. Specific Embodiments of Anti-CD22 Antibodies

In one aspect, the invention provides an antibody comprising at leastone, two, three, four, five, or six HVRs selected from (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO:2; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO:4; (c) an HVR-H3comprising an amino acid sequence selected from SEQ ID NO:6; (d) anHVR-L1 comprising the amino acid sequence of any one of SEQ ID NO:9, 10,19, 20, 21, 22, 23; (e) an HVR-L2 comprising the amino acid sequence ofSEQ ID NO:12; and (f) an HVR-L3 comprising an amino acid sequenceselected from SEQ ID NO:14.

In one aspect, the invention provides an anti-CD22 antibody comprisingat least one, at least two, or all three VH HVR sequences selected from(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) an HVR-H3comprising an amino acid sequence selected from SEQ ID NO:6. In oneaspect, the invention provides an anti-CD22 antibody comprising anHVR-H1 comprising the amino acid sequence of SEQ ID NO:2. In one aspect,the invention provides an anti-CD22 antibody comprising an HVR-H2comprising the amino acid sequence of SEQ ID NO:4. In one aspect, theinvention provides an anti-CD22 antibody comprising an HVR-H3 comprisingan amino acid sequence selected from SEQ ID NO:6.

In one aspect, the invention provides an anti-CD22 antibody comprisingan HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:6and an HVR-H1 comprising an amino acid sequence selected from SEQ IDNO:2.

In one aspect, the invention provides an anti-CD22 antibody comprisingan HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:6and an HVR-H2 comprising an amino acid sequence selected from SEQ IDNO:4.

In one aspect, the invention provides an anti-CD22 antibody comprisingan HVR-H1 comprising the amino acid sequence of SEQ ID NO:2 and anHVR-H2 comprising the amino acid sequence of SEQ ID NO:4.

In one aspect, the invention provides an anti-CD22 antibody comprisingan HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; an HVR-H2comprising the amino acid sequence of SEQ ID NO:4; and an HVR-H3comprising the amino acid sequence of SEQ ID NO:6.

In one aspect, the invention provides an anti-CD22 antibody comprisingat least one, at least two, or all three VL HVR sequences selected from(a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:9 or SEQID NO:10; (b) an HVR-L2 comprising the amino acid sequence of SEQ IDNO:12; and (c) an HVR-L3 comprising an amino acid sequence selected fromSEQ ID NO:14. In one aspect, the invention provides an anti-CD22antibody comprising an HVR-L1 comprising an amino acid sequence selectedfrom SEQ ID NO:9. In one aspect, the invention provides an anti-CD22antibody comprising an HVR-L1 comprising an amino acid sequence selectedfrom SEQ ID NO:10. In one aspect, the invention provides an anti-CD22antibody comprising an HVR-L1 comprising an amino acid sequence selectedfrom SEQ ID NO:19-23. In one aspect, the HVR-L1 comprises the amino acidsequence of SEQ ID NO:9 wherein N28 is replaced by V (an N28V amino acidchange, which generates SEQ ID NO:10). In one aspect, the HVR-L1comprises the amino acid sequence of SEQ ID NO:9 wherein N28 is replacedby A (an N28A amino acid change, which generates SEQ ID NO:19). In oneaspect, the HVR-L1 comprises the amino acid sequence of SEQ ID NO:9wherein N28 is replaced by Q (an N28Q amino acid change, which generatesSEQ ID NO:20). In one aspect, the HVR-L1 comprises the amino acidsequence of SEQ ID NO:9 wherein N28 is replaced by S (an N28S amino acidchange, which generates SEQ ID NO:21). In one aspect, the HVR-L1comprises the amino acid sequence of SEQ ID NO:9 wherein N28 is replacedby D (an N28D amino acid change, which generates SEQ ID NO:22). In oneaspect, the HVR-L1 comprises the amino acid sequence of SEQ ID NO:9wherein N28 is replaced by I (an N28I amino acid change, which generatesSEQ ID NO:23). In one aspect, the invention provides an anti-CD22antibody comprising an HVR-L1 comprising the amino acid sequence of anyone of SEQ ID NO:9, 10, 19, 20, 21, 22, 23. In one aspect, the HVR-L1 isany one of SEQ ID NO:9, 10, 19, 20, 21, 22, or 23 and the amino acid atposition N30 (asparagine at position 30) is replaced by A (an N30A aminoacid change). In one aspect, the HVR-L1 is any one of SEQ ID NO:9, 10,19, 20, 21, 22, or 23 and the amino acid at position N30 (asparagine atposition 30) is replaced by Q (an N30Q amino acid change).

In one aspect, the invention provides an anti-CD22 antibody comprising(a) an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6 and (b)an HVR-L3 comprising an amino acid sequence of SEQ ID NO:14. In someembodiments, the CD22 antibody further comprises (a) an HVR-H1comprising SEQ ID NO:2 and an HVR-H2 comprising SEQ ID NO:4.

In one aspect, the invention provides an anti-CD22 antibody comprising(a) an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6 and (b)an HVR-L2 comprising an amino acid sequence of SEQ ID NO:12. In someembodiments, the CD22 antibody further comprises (a) an HVR-H1comprising SEQ ID NO:2 and an HVR-H2 comprising SEQ ID NO:4.

In one aspect, the invention provides an anti-CD22 antibody comprising(a) an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6 and (b)an HVR-L1 comprising an amino acid sequence selected from SEQ ID NO:9,10, 19, 20, 21, 22, and 23. In some embodiments, the CD22 antibodyfurther comprises (a) an HVR-H1 comprising SEQ ID NO:2 and an HVR-H2comprising SEQ ID NO:4. In some embodiments, the amino acid sequence ofSEQ ID NO:9, 10, 19, 20, 21, 22, or 23 comprises an N30A or N30Q aminoacid change. In some embodiments, the CD22 antibody further comprisesHVR-L2 comprising the amino acid sequence of SEQ ID NO:12. In someembodiments, the CD22 antibody further comprises HVR-L3 comprising theamino acid sequence of SEQ ID NO:14.

In one aspect, the invention provides an anti-CD22 antibody comprising(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:6; (d) an HVR-L1comprising the amino acid sequence selected from SEQ ID NO:9, 10, 19,20, 21, 22, 23; (e) an HVR-L2 comprising the amino acid sequence of SEQID NO:12; and an HVR-L3 comprising the amino acid sequence of SEQ IDNO:14. In some embodiments, the invention further provides that theamino acid sequence SEQ ID NO:9, 10, 19, 20, 21, 22, or 23 selected asHVR-L1 is modified by an N30A or an N30Q amino acid change.

In one aspect, the invention provides an anti-CD22 antibody comprising aheavy chain variable domain comprising SEQ ID NO:16 (see FIG. 2A,h10F4v1). In one aspect, the invention provides an anti-CD22 antibodycomprising a light chain variable domain comprising SEQ ID NO:17 (seeFIG. 2B, h10F4v1). In one aspect, the invention provides an anti-CD22antibody comprising a light chain variable domain comprising SEQ IDNO:18 (see FIG. 2B, h10F4v3).

In one aspect, the invention provides an anti-CD22 antibody comprising aheavy chain comprising SEQ ID NO:34 (see FIG. 2A, m10F4). In one aspect,the invention provides an anti-CD22 antibody comprising a light chaincomprising SEQ ID NO:35 (see FIG. 2B, m10F4).

In one aspect, the invention provides an anti-CD22 antibody comprising1, 2, 3, 4, 5, or 6 of the HVR sequences of the antibody 10F4.4.1produced by the hybridoma deposited with the ATCC and having accessionnumber PTA-7621.

In one aspect, the invention provides an anti-CD22 antibody comprising1, 2, 3, 4, 5, or 6 of the HVR sequences of the antibody 5E8.1.8produced by the hybridoma deposited with the ATCC and having accessionnumber PTA-7620.

An anti-CD22 antibody may comprise any suitable framework variabledomain sequence, provided that the antibody retains the ability to bindCD22. For example, in some embodiments, anti-CD22 antibodies of theinvention comprise a human subgroup III heavy chain framework consensussequence. In one embodiment of these antibodies, the heavy chainframework consensus sequence comprises substitution(s) at position 71,73 and/or 78. In one embodiments of these antibodies, position 71 is A,position 73 is T, and/or position 78 is A. In one embodiment, theseantibodies comprise a heavy chain variable domain framework sequence ofhuMAb4D5-8, e.g., SEQ ID NO:1, 3, 5, 7 (FR-H1, FR-H2, FR-H3, FR-H4,respectively). huMAb4D5-8 is commercially known as HERCEPTIN® anti-HER2antibody, Genentech, Inc., South San Francisco, Calif., USA; alsoreferred to in U.S. Pat. Nos. 6,407,213 & 5,821,337, and Lee et al., J.Mol. Biol. (2004), 340(5):1073-93. In one such embodiment, theseantibodies further comprise a human I light chain framework consensussequence. In one such embodiment, these antibodies comprise a lightchain variable domain framework sequence of huMAb4D5-8, e.g. SEQ IDNO:8, 1, 13, 15 (FR-L1, FR-L2, FR-L3, FR-L4, respectively).

In one embodiment, an anti-CD22 antibody comprises a heavy chainvariable domain comprising a framework sequence and hypervariableregions, wherein the framework sequence comprises the FR-H1-FR-H4sequences SEQ ID NO:1, 3, 5, and 7, respectively; the HVR H1 comprisesthe amino acid sequence of SEQ ID NO:2; the HVR-H2 comprises the aminoacid sequence of SEQ ID NO:4; and the HVR-H3 comprises an amino acidsequence selected from SEQ ID NO:6. In one embodiment, an anti-CD22antibody comprises a light chain variable domain comprising a frameworksequence and hypervariable regions, wherein the framework sequencecomprises the FR-L1-FR-L4 sequences of SEQ ID NO:8, 11, 13, and 15,respectively; the HVR-L1 comprises the amino acid sequence selected fromSEQ ID NO:9, 10, 19, 20, 21, 22, and 23, wherein any one of SEQ IDNOS:9-10 or 19-23 may comprise a N30A or N30Q amino acid change; theHVR-L2 comprises the amino acid sequence of SEQ ID NO:12; and the HVR-L3comprises an amino acid sequence selected from SEQ ID NO:14. In oneembodiment of these antibodies, the heavey chain variable domaincomprises SEQ ID NO:16 and the light chain variable domain comprises SEQID NO:17 or 18.

In some embodiments, the invention provides an anti-CD22 antibodycomprising a heavy chain variable domain comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to an amino acid sequence SEQ ID NO:16. In someembodiments, an amino acid sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity containssubstitutions, insertions, or deletions relative to the referencesequence, but an antibody comprising that amino acid sequence retainsthe ability to bind to CD22. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, or deleted in a sequenceSEQ ID NO:16. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs). In someembodiments, an anti-CD22 antibody comprises a heavy chain variabledomain comprising an amino acid sequence selected from SEQ ID NO:16.

In some embodiments, the invention provides an anti-CD22 antibodycomprising a heavy chain variable domain as depicted in below.

(SEQ ID NO: 16) 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Glu Phe Ser Arg Ser Trp Met AsnTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Tyr ProGly Asp Gly Asp Thr Asn Tyr Ser Gly Lys Phe Lys Gly Lys Ala Thr Leu ThrAla Asp Lys Ser Ser Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala GluAsp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gly Ser Ser Trp Asp Trp Tyr PheAsp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 113 (HVR residues are underlined).

In some embodiments, the heavy chain HVR and FR sequences comprise thefollowing:

HVR-H1 (Gly Tyr Glu Phe Ser Arg Ser Trp Met Asn, SEQ ID NO: 2) HVR-H2(Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Ser Gly Lys PheLys Gly, SEQ ID NO: 4) HVR-H3(Asp Gly Ser Ser Trp Asp Try Tyr Phe Asp Tyr, SEQ ID NO: 6) FR-H1(Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser, SEQ ID NO: 1) FR-H2(Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val, SEQ ID NO: 3)FR-H3 (Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Leu GlnMet Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg, SEQ ID NO: 5)FR-H4 (Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser, SEQ ID NO: 7)

In some embodiments, the invention provides an anti-CD22 antibodycomprising a light chain variable domain as depicted in below.

(SEQ ID NO: 17) 1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His Ser AsnGly Asn Thr Phe Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala ProLys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro SerArg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly SerGln Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 108 (HVR residues are underlined and position N28 is in bold type) Or(SEQ ID NO: 18) 1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His Ser ValGly Asn Thr Phe Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala ProLys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro SerArg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly SerGln Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 108(HVR residues are underlined and position N28V is in bold type).

In some embodiments, the light chain HVR sequences comprise thefollowing:

HVR-L1 (Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly AsnThr Phe Leu Glu, SEQ ID NO: 9) HVR-L1(Arg Ser Ser Gln Ser Ile Val His Ser Val Gly AsnThr Phe Leu Glu, SEQ ID NO: 10) HVR-L1(Arg Ser Ser Gln Ser Ile Val His Ser Ala Gly AsnThr Phe Leu Glu, SEQ ID NO: 19) HVR-L1(Arg Ser Ser Gln Ser Ile Val His Ser Gln Gly AsnThr Phe Leu Glu, SEQ ID NO: 20) HVR-L1(Arg Ser Ser Gln Ser Ile Val His Ser Ser Gly AsnThr Phe Leu Glu, SEQ ID NO: 21) HVR-L1(Arg Ser Ser Gln Ser Ile Val His Ser Asp Gly AsnThr Phe Leu Glu, SEQ ID NO: 22) HVR-L1(Arg Ser Ser Gln Ser Ile Val His Ser Ile Gly AsnThr Phe Leu Glu, SEQ ID NO: 23) HVR-L1(Arg Ser Ser Gln Ser Ile Val His Ser Ile Gly AlaThr Phe Leu Glu, SEQ ID NO: 32) HVR-L1(Arg Ser Ser Gln Ser Ile Val His Ser Ile Gly GlnThr Phe Leu Glu, SEQ ID NO: 33) HVR-L2(Lys Val Ser Asn Arg Phe Ser, SEQ ID NO: 12) HVR-L3(Phe Gln Gly Ser Gln Phe Pro Tyr Thr, SEQ ID NO: 14).

In some embodiments, the light chain FR sequences comprise thefollowing:

FR-L1 (Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys, SEQ ID NO: 8); FR-L2(Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys LeuLeu Ile Tyr, SEQ ID NO: 11); FR-L3(Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser GlyThr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln ProGlu Asp Phe Ala Thr Tyr Tyr Cys, SEQ ID NO: 13) FR-L4(Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg, SEQ ID NO: 15).

In one aspect, the invention provides an anti-CD22 antibody comprising alight chain variable domain comprising an amino acid sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to an amino acid sequence selected from SEQ ID NO:17 or 18. Insome embodiments, an amino acid sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity containssubstitutions, additions, or deletions relative to the referencesequence, but an antibody comprising that amino acid sequence retainsthe ability to bind to CD22. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, or deleted in a sequenceselected from SEQ ID NO:17 or 18. In some embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). In some embodiments, an anti-CD22 antibodycomprises a light chain variable domain comprising an amino acidsequence selected from SEQ ID NO:17 or 18.

In one aspect, the invention provides an anti-CD22 antibody comprising(a) a heavy chain variable domain comprising an amino acid sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to an amino acid sequence selected from SEQ ID NO:16;and (b) a light chain variable domain comprising an amino acid sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to an amino acid sequence selected from SEQ ID NO:17or 18. In some embodiments, an amino acid sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identitycontains substitutions, additions, or deletions relative to thereference sequence, but an antibody comprising that amino acid sequenceretains the ability to bind to CD22. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, or deleted in thereference sequence. In some embodiments, the substitutions, insertions,or deletions occur in regions outside the HVRs (i.e., in the FRs). Insome embodiments, an anti-CD22 antibody comprises a heavy chain variabledomain comprising an amino acid sequence of SEQ ID NO:16 and a lightchain variable domain comprising an amino acid sequence selected fromSEQ ID NO:18.

In one aspect, the invention provides an anti-CD22 antibody comprising(a) one, two, or three VH HVRs selected from those shown in FIG. 2Aand/or (b) one, two, or three VL HVRs selected from those shown in FIG.2B. In one aspect, the invention provides an anti-CD22 antibodycomprising a heavy chain variable domain selected from those shown inFIG. 2A and a light chain variable domain selected from those shown inFIG. 2B.

In one aspect, the anti-CD22 antibody of the invention comprises 1, 2,3, 4, 5, or 6 of the hypervariable regions of the 5E8.1.8 antibodyproduced by the hybridoma deposited with the ATCC and having accessionno. PTA-7620.

2. Antibody Fragments

The present invention encompasses antibody fragments. Antibody fragmentsmay be generated by traditional means, such as enzymatic digestion, orby recombinant techniques. In certain circumstances there are advantagesof using antibody fragments, rather than whole antibodies. The smallersize of the fragments allows for rapid clearance, and may lead toimproved access to solid tumors. For a review of certain antibodyfragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

3. Humanized Antibodies

The invention encompasses humanized antibodies. Various methods forhumanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human.

These non-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al.(1988) Nature 332:323-327; Verhoeyen et al. (1988) Science239:1534-1536), by substituting hypervariable region sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further generally desirable that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to one method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

4. Human Antibodies

Human anti-CD22 antibodies of the invention can be constructed bycombining Fv clone variable domain sequence(s) selected fromhuman-derived phage display libraries with known human constant domainsequences(s) as described above. Alternatively, human monoclonalanti-CD22 antibodies of the invention can be made by the hybridomamethod. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described, forexample, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol.,147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

5. Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different antigens. In certainembodiments, bispecific antibodies are human or humanized antibodies. Incertain embodiments, one of the binding specificities is for CD22 andthe other is for any other antigen. In certain embodiments, bispecificantibodies may bind to two different epitopes of CD22. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress CD22. These antibodies possess a CD22-binding arm and an armwhich binds a cytotoxic agent, such as, e.g., saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion, forexample, is with an immunoglobulin heavy chain constant domain,comprising at least part of the hinge, CH2, and CH3 regions. In certainembodiments, the first heavy-chain constant region (CH1), containing thesite necessary for light chain binding, is present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The interfacecomprises at least a part of the C_(H)3 domain of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). This providesa mechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking method. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

6. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. In certain embodiments, the dimerization domain comprises(or consists of) an Fc region or a hinge region. In this scenario, theantibody will comprise an Fc region and three or more antigen bindingsites amino-terminal to the Fc region. In certain embodiments, amultivalent antibody comprises (or consists of) three to about eightantigen binding sites. In one such embodiment, a multivalent antibodycomprises (or consists of) four antigen binding sites. The multivalentantibody comprises at least one polypeptide chain (for example, twopolypeptide chains), wherein the polypeptide chain(s) comprise two ormore variable domains. For instance, the polypeptide chain(s) maycomprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein may further comprise atleast two (for example, four) light chain variable domain polypeptides.The multivalent antibody herein may, for instance, comprise from abouttwo to about eight light chain variable domain polypeptides. The lightchain variable domain polypeptides contemplated here comprise a lightchain variable domain and, optionally, further comprise a CL domain.

7. Single-Domain Antibodies

In some embodiments, an antibody of the invention is a single-domainantibody. A single-domain antibody is a single polypeptide chaincomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).In one embodiment, a single-domain antibody consists of all or a portionof the heavy chain variable domain of an antibody.

8. Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (e.g.,alanine or polyalanine) to affect the interaction of the amino acidswith antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site, alascanning or random mutagenesis is conducted at the target codon orregion and the expressed immunoglobulins are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

In certain embodiments, an antibody of the invention is altered toincrease or decrease the extent to which the antibody is glycosylated.Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of a carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites) is created or removed. The alteration may also bemade by the addition, deletion, or substitution of one or more serine orthreonine residues to the sequence of the original antibody (forO-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with abisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached toan Fc region of the antibody are referenced in WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO 1997/30087, Patel et al.See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof. See also US 2005/0123546 (Umana et al.) onantigen-binding molecules with modified glycosylation.

In certain embodiments, a glycosylation variant comprises an Fc region,wherein a carbohydrate structure attached to the Fc region lacks fucose.Such variants have improved ADCC function. Optionally, the Fc regionfurther comprises one or more amino acid substitutions therein whichfurther improve ADCC, for example, substitutions at positions 298, 333,and/or 334 of the Fc region (Eu numbering of residues). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodiesinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-glucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

In one embodiment, the antibody is altered to improve its serumhalf-life. To increase the serum half life of the antibody, one mayincorporate a salvage receptor binding epitope into the antibody(especially an antibody fragment) as described in U.S. Pat. No.5,739,277, for example. As used herein, the term “salvage receptorbinding epitope” refers to an epitope of the Fc region of an IgGmolecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible forincreasing the in vivo serum half-life of the IgG molecule (US2003/0190311, U.S. Pat. No. 6,821,505; U.S. Pat. No. 6,165,745; U.S.Pat. No. 5,624,821; U.S. Pat. No. 5,648,260; U.S. Pat. No. 6,165,745;U.S. Pat. No. 5,834,597).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. Sites of interest for substitutionalmutagenesis include the hypervariable regions, but FR alterations arealso contemplated. Conservative substitutions are shown in Table 1 underthe heading of “preferred substitutions.” If such substitutions resultin a desirable change in biological activity, then more substantialchanges, denominated “exemplary substitutions” in Table 1, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (O)

(3) acidic: Asp (D), Glu (E) (4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: H is, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, into theremaining (non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have modified (e.g., improved) biologicalproperties relative to the parent antibody from which they aregenerated. A convenient way for generating such substitutional variantsinvolves affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino acid substitutions at each site. The antibodies thusgenerated are displayed from filamentous phage particles as fusions toat least part of a phage coat protein (e.g., the gene III product ofM13) packaged within each particle. The phage-displayed variants arethen screened for their biological activity (e.g. binding affinity). Inorder to identify candidate hypervariable region sites for modification,scanning mutagenesis (e.g., alanine scanning) can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and antigen. Such contact residuesand neighboring residues are candidates for substitution according totechniques known in the art, including those elaborated herein. Oncesuch variants are generated, the panel of variants is subjected toscreening using techniques known in the art, including those describedherein, and antibodies with superior properties in one or more relevantassays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of antibodies of the invention, thereby generating an Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions including that of a hinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody of the invention maycomprise one or more alterations as compared to the wild typecounterpart antibody, e.g. in the Fc region. These antibodies wouldnonetheless retain substantially the same characteristics required fortherapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants. WO00/42072(Presta) and WO 2004/056312 (Lowman) describe antibody variants withimproved or diminished binding to FcRs. The content of these patentpublications are specifically incorporated herein by reference. See,also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodieswith increased half lives and improved binding to the neonatal Fcreceptor (FcRn), which is responsible for the transfer of maternal IgGsto the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al.,J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton etal.). These antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1, WO99/51642. The contents of those patent publicationsare specifically incorporated herein by reference. See, also, Idusogieet al. J. Immunol. 164: 4178-4184 (2000).

In one aspect, the invention provides antibodies comprisingmodifications in the interface of Fc polypeptides comprising the Fcregion, wherein the modifications facilitate and/or promoteheterodimerization. These modifications comprise introduction of aprotuberance into a first Fc polypeptide and a cavity into a second Fcpolypeptide, wherein the protuberance is positionable in the cavity soas to promote complexing of the first and second Fc polypeptides.Methods of generating antibodies with these modifications are known inthe art, e.g., as described in U.S. Pat. No. 5,731,168.

9. Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. 102: 11600-11605 (2005)).The radiation may be of any wavelength, and includes, but is not limitedto, wavelengths that do not harm ordinary cells, but which heat thenonproteinaceous moiety to a temperature at which cells proximal to theantibody-nonproteinaceous moiety are killed.

Certain Methods of Making Antibodies

1. Certain Hybridoma-Based Methods

The anti-CD22 monoclonal antibodies of the invention can be made usingthe hybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Antibodies to CD22 generally are raisedin animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of CD22 and an adjuvant. CD22 may be prepared using methodswell-known in the art, some of which are further described herein. Forexample, CD22 may be produced recombinantly. In one embodiment, animalsare immunized with a derivative of CD22 that contains an extracellularportion of CD22 fused to the Fc portion of an immunoglobulin heavychain. In one embodiment, animals are immunized with an CD22-IgG1 fusionprotein. In one embodiment, animals are immunized with immunogenicderivatives of CD22 in a solution with monophosphoryl lipid A(MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem. Research, Inc.,Hamilton, Mont.), and the solution is injected intradermally at multiplesites. Two weeks later the animals are boosted. Seven to fourteen dayslater the animals are bled, and the serum is assayed for anti-CD22titer. Animals are boosted until titer plateaus.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium, e.g., a medium that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

In certain embodiments, myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Exemplary myeloma cells include, but are not limited to, murinemyeloma lines, such as those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies that bind to CD22. Preferably, thebinding specificity of monoclonal antibodies produced by hybridoma cellsis determined by immunoprecipitation or by an in vitro binding assay,such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay(ELISA). The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson et al., Anal.Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.Monoclonal antibodies secreted by the subclones are suitably separatedfrom the culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

2. Certain Library Screening Methods

Anti-CD22 antibodies of the invention can be made by using combinatoriallibraries to screen for antibodies with the desired activity oractivities. For example, a variety of methods are known in the art forgenerating phage display libraries and screening such libraries forantibodies possessing the desired binding characteristics. Such methodsare described generally in Hoogenboom et al. (2001) in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J.), and in certain embodiments, in Lee et al. (2004) J. Mol. Biol.340:1073-1093.

In principle, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the non-bindingclones in the library. The binding clones are then eluted from theantigen, and can be further enriched by additional cycles of antigenadsorption/elution. Any of the anti-CD22 antibodies of the invention canbe obtained by designing a suitable antigen screening procedure toselect for the phage clone of interest followed by construction of afull length anti-CD22 antibody clone using the Fv sequences from thephage clone of interest and suitable constant region (Fc) sequencesdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991),vols. 1-3.

In certain embodiments, the antigen-binding domain of an antibody isformed from two variable (V) regions of about 110 amino acids, one eachfrom the light (VL) and heavy (VH) chains, that both present threehypervariable loops (HVRs) or complementarity-determining regions(CDRs). Variable domains can be displayed functionally on phage, eitheras single-chain Fv (scFv) fragments, in which VH and VL are covalentlylinked through a short, flexible peptide, or as Fab fragments, in whichthey are each fused to a constant domain and interact non-covalently, asdescribed in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Asused herein, scFv encoding phage clones and Fab encoding phage clonesare collectively referred to as “Fv phage clones” or “Fv clones.”

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

In certain embodiments, filamentous phage is used to display antibodyfragments by fusion to the minor coat protein pIII. The antibodyfragments can be displayed as single chain Fv fragments, in which VH andVL domains are connected on the same polypeptide chain by a flexiblepolypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol.,222: 581-597 (1991), or as Fab fragments, in which one chain is fused topIII and the other is secreted into the bacterial host cell periplasmwhere assembly of a Fab-coat protein structure which becomes displayedon the phage surface by displacing some of the wild type coat proteins,e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137(1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-CD22 clones is desired, the subject is immunized withCD22 to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In a preferred embodiment, a humanantibody gene fragment library biased in favor of anti-CD22 clones isobtained by generating an anti-CD22 antibody response in transgenic micecarrying a functional human immunoglobulin gene array (and lacking afunctional endogenous antibody production system) such that CD22immunization gives rise to B cells producing human antibodies againstCD22. The generation of human antibody-producing transgenic mice isdescribed below.

Additional enrichment for anti-CD22 reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing CD22-specific membrane bound antibody, e.g., by cellseparation using CD22 affinity chromatography or adsorption of cells tofluorochrome-labeled CD22 followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which CD22 isnot antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). In certain embodiments, library diversity is maximized by usingPCR primers targeted to each V-gene family in order to amplify allavailable VH and VL arrangements present in the immune cell nucleic acidsample, e.g. as described in the method of Marks et al., J. Mol. Biol.,222: 581-597 (1991) or as described in the method of Orum et al.,Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplifiedDNA into expression vectors, rare restriction sites can be introducedwithin the PCR primer as a tag at one end as described in Orlandi et al.(1989), or by further PCR amplification with a tagged primer asdescribed in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanV_(κ) and Vλ segments have been cloned and sequenced (reported inWilliams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can beused to make synthetic light chain repertoires. Synthetic V generepertoires, based on a range of VH and VL folds, and L3 and H3 lengths,will encode antibodies of considerable structural diversity. Followingamplification of V-gene encoding DNAs, germline V-gene segments can berearranged in vitro according to the methods of Hoogenboom and Winter,J. Mol. Biol., 227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (k_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci. USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities of about 10⁻⁹ M or less.

Screening of the libraries can be accomplished by various techniquesknown in the art. For example, CD22 can be used to coat the wells ofadsorption plates, expressed on host cells affixed to adsorption platesor used in cell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other method for panning phagedisplay libraries.

The phage library samples are contacted with immobilized CD22 underconditions suitable for binding at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by CD22 antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1.000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for CD22.However, random mutation of a selected antibody (e.g. as performed insome affinity maturation techniques) is likely to give rise to manymutants, most binding to antigen, and a few with higher affinity. Withlimiting CD22, rare high affinity phage could be competed out. To retainall higher affinity mutants, phages can be incubated with excessbiotinylated CD22, but with the biotinylated CD22 at a concentration oflower molarity than the target molar affinity constant for CD22. Thehigh affinity-binding phages can then be captured by streptavidin-coatedparamagnetic beads. Such “equilibrium capture” allows the antibodies tobe selected according to their affinities of binding, with sensitivitythat permits isolation of mutant clones with as little as two-foldhigher affinity from a great excess of phages with lower affinity.Conditions used in washing phages bound to a solid phase can also bemanipulated to discriminate on the basis of dissociation kinetics.

Anti-CD22 clones may be selected based on activity. In certainembodiments, the invention provides anti-CD22 antibodies that bind toliving cells that naturally express CD22. In one embodiment, theinvention provides anti-CD22 antibodies that block the binding between aCD22 ligand and CD22, but do not block the binding between a CD22 ligandand a second protein. Fv clones corresponding to such anti-CD22antibodies can be selected by (1) isolating anti-CD22 clones from aphage library as described above, and optionally amplifying the isolatedpopulation of phage clones by growing up the population in a suitablebacterial host; (2) selecting CD22 and a second protein against whichblocking and non-blocking activity, respectively, is desired; (3)adsorbing the anti-CD22 phage clones to immobilized CD22; (4) using anexcess of the second protein to elute any undesired clones thatrecognize CD22-binding determinants which overlap or are shared with thebinding determinants of the second protein; and (5) eluting the cloneswhich remain adsorbed following step (4). Optionally, clones with thedesired blocking/non-blocking properties can be further enriched byrepeating the selection procedures described herein one or more times.

DNA encoding hybridoma-derived monoclonal antibodies or phage display Fvclones of the invention is readily isolated and sequenced usingconventional procedures (e.g. by using oligonucleotide primers designedto specifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of antibody-encoding DNA includeSkerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun,Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. An Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In certain embodiments,an Fv clone derived from human variable DNA is fused to human constantregion DNA to form coding sequence(s) for full- or partial-length humanheavy and/or light chains.

DNA encoding anti-CD22 antibody derived from a hybridoma of theinvention can also be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofhomologous murine sequences derived from the hybridoma clone (e.g. as inthe method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855(1984)). DNA encoding a hybridoma- or Fv clone-derived antibody orfragment can be further modified by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of the Fvclone or hybridoma clone-derived antibodies of the invention.

3. Vectors, Host Cells, and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, hostcells are of either prokaryotic or eukaryotic (generally mammalian)origin. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

Generating Antibodies Using Prokaryotic Host Cells:

Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Antibodies of the invention can also be produced by using an expressionsystem in which the quantitative ratio of expressed polypeptidecomponents can be modulated in order to maximize the yield of secretedand properly assembled antibodies of the invention. Such modulation isaccomplished at least in part by simultaneously modulating translationalstrengths for the polypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence. In certain embodiments, changes in the nucleotidesequence are silent. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

In one embodiment, a set of vectors is generated with a range of TIRstrengths for each cistron therein. This limited set provides acomparison of expression levels of each chain as well as the yield ofthe desired antibody products under various TIR strength combinations.TIR strengths can be determined by quantifying the expression level of areporter gene as described in detail in Simmons et al. U.S. Pat. No.5,840,523. Based on the translational strength comparison, the desiredindividual TIRs are selected to be combined in the expression vectorconstructs of the invention.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coli, 1776 (ATCC31,537) and E. coli RV308 (ATCC 31,608) are also suitable. Theseexamples are illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. Incertain embodiments, for E. coli growth, growth temperatures range fromabout 20° C. to about 39° C.; from about 25° C. to about 37° C.; orabout 30° C. The pH of the medium may be any pH ranging from about 5 toabout 9, depending mainly on the host organism. In certain embodiments,for E. coli, the pH is from about 6.8 to about 7.4, or about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. In certain embodiments, thephosphate-limiting medium is the C.R.A.P. medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, and in certain embodiments, about 1,000 to 100,000 liters ofcapacity. These fermentors use agitator impellers to distribute oxygenand nutrients, especially glucose (the preferred carbon/energy source).Small scale fermentation refers generally to fermentation in a fermentorthat is no more than approximately 100 liters in volumetric capacity,and can range from about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

Antibody Purification

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the antibody products of the invention.Protein A is a 41kD cell wall protein from Staphylococcus aureas whichbinds with a high affinity to the Fc region of antibodies. Lindmark etal (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein Ais immobilized can be a column comprising a glass or silica surface, ora controlled pore glass column or a silicic acid column. In someapplications, the column is coated with a reagent, such as glycerol, topossibly prevent nonspecific adherence of contaminants.

As the first step of purification, a preparation derived from the cellculture as described above can be applied onto a Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase would then be washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

Generating Antibodies Using Eukaryotic Host Cells:

A vector for use in a eukaryotic host cell generally includes one ormore of the following non-limiting components: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected may be one that is recognized andprocessed (i.e., cleaved by a signal peptidase) by the host cell. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such a precursor region is ligated in readingframe to DNA encoding the antibody.

Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, in some embodiments, cells transformed with the DHFRselection gene are first identified by culturing all of thetransformants in a culture medium that contains methotrexate (Mtx), acompetitive antagonist of DHFR. In some embodiments, an appropriate hostcell when wild-type DHFR is employed is the Chinese hamster ovary (CHO)cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding a polypeptide of interest (e.g., an antibody). Promotersequences are known for eukaryotes. For example, virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. In certain embodiments, any or all of these sequences may besuitably inserted into eukaryotic expression vectors.

Transcription from vectors in mammalian host cells is controlled, forexample, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, from heat-shock promoters, provided such promoters arecompatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982), describingexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

Enhancer Element Component

Transcription of DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) describing enhancerelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the antibodypolypeptide-encoding sequence, but is generally located at a site 5′from the promoter.

Transcription Termination Component

Expression vectors used in eukaryotic host cells may also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/−DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othersupplements may also be included at appropriate concentrations thatwould be known to those skilled in the art. The culture conditions, suchas temperature, pH, and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, may be removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems may be firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis, and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a convenient technique. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc domain that is present in the antibody. Protein A can be used topurify antibodies that are based on human γ1, γ2, or γ4 heavy chains(Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached may be agarose, but other matrices are available. Mechanicallystable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to furtherpurification, for example, by low pH hydrophobic interactionchromatography using an elution buffer at a pH between about 2.5-4.5,preferably performed at low salt concentrations (e.g., from about0-0.25M salt).

In general, various methodologies for preparing antibodies for use inresearch, testing, and clinical use are well-established in the art,consistent with the above-described methodologies and/or as deemedappropriate by one skilled in the art for a particular antibody ofinterest.

Immunoconjugates

The invention also provides immunoconjugates (interchangeably referredto as “antibody-drug conjugates,” or “ADCs”) comprising any of theanti-CD22 antibodies of the invention conjugated to one or morecytotoxic agents, such as a chemotherapeutic agent, a drug, a growthinhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

In certain embodiments, an immunoconjugate comprises an anti-CD22antibody and a chemotherapeutic agent or other toxin. Chemotherapeuticagents useful in the generation of immunoconjugates are described herein(e.g., above). Enzymatically active toxins and fragments thereof canalso be used and are described herein.

In certain embodiments, an immunoconjugate comprises an anti-CD22antibody and one or more small molecule toxins, including, but notlimited to, small molecule drugs such as a calicheamicin, maytansinoid,dolastatin, auristatin, trichothecene, and CC1065, and the derivativesof these drugs that have cytotoxic activity. Examples of suchimmunoconjugates are discussed in further detail below.

1. Exemplary Immunoconjugates—Antibody Drug Conjugates

An immunoconjugate (or “antibody-drug conjugate” (“ADC”)) of theinvention may be of Formula I, below, wherein an anti-CD22 antibody isconjugated (i.e., covalently attached) to one or more drug moieties (D)through an optional linker (L).

Ab-(L-D)_(p)  Formula I

Accordingly, the anti-CD22 antibody may be conjugated to the drug eitherdirectly or via a linker. In Formula I, p is the average number of drugmoieties per antibody, which can range, e.g., from about 1 to about 20drug moieties per antibody, and in certain embodiments, from 1 to about8 drug moieties per antibody.

Exemplary Linkers

Exemplary linkers and drug moieties are disclosed herein. A linker maycomprise one or more linker components. Exemplary linker componentsinclude 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl 4-(2pentanoate (“SPP”), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate (“SMCC”), and N-Succinimidyl (4-iodo-acetyl) aminobenzoate(“SIAB”). Various linker components are known in the art, some of whichare described below.

A linker may be a “cleavable linker,” facilitating release of a drug inthe cell. For example, an acid-labile linker (e.g., hydrazone),protease-sensitive (e.g., peptidase-sensitive) linker, photolabilelinker, dimethyl linker or disulfide-containing linker (Chari et al.,Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.

In some embodiments, a linker component may comprise a “stretcher unit”that links an antibody to another linker component or to a drug moiety.Exemplary stretcher units are shown below (wherein the wavy lineindicates sites of covalent attachment to an antibody):

In some embodiments, a linker component may comprise an amino acid unit.In one such embodiment, the amino acid unit allows for cleavage of thelinker by a protease, thereby facilitating release of the drug from theimmunoconjugate upon exposure to intracellular proteases, such aslysosomal enzymes. See, e.g., Doronina et al. (2003) Nat. Biotechnol.21:778-784. Exemplary amino acid units include, but are not limited to,a dipeptide, a tripeptide, a tetrapeptide, and a pentapeptide. Exemplarydipeptides include: valine-citrulline (vc or val-cit),alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk orphe-lys); or N-methyl-valine-citrulline (Me-val-cit). Exemplarytripeptides include: glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally, as well as minor amino acidsand non-naturally occurring amino acid analogs, such as citrulline.Amino acid units can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzyme, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

In some embodiments, a linker component may comprise a “spacer” unitthat links the antibody to a drug moiety, either directly or by way of astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon enzymatic (e.g., proteolytic) cleavage of theADC. Examples of non-self-immolative spacer units include, but are notlimited to, a glycine spacer unit and a glycine-glycine spacer unit.Other combinations of peptidic spacers susceptible to sequence-specificenzymatic cleavage are also contemplated. For example, enzymaticcleavage of an ADC containing a glycine-glycine spacer unit by atumor-cell associated protease would result in release of aglycine-glycine-drug moiety from the remainder of the ADC. In one suchembodiment, the glycine-glycine-drug moiety is then subjected to aseparate hydrolysis step in the tumor cell, thus cleaving theglycine-glycine spacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moietywithout a separate hydrolysis step. In certain embodiments, a spacerunit of a linker comprises a p-aminobenzyl unit. In one such embodiment,a p-aminobenzyl alcohol is attached to an amino acid unit via an amidebond, and a carbamate, methylcarbamate, or carbonate is made between thebenzyl alcohol and a cytotoxic agent. See, e.g., Hamann et al. (2005)Expert Opin. Ther. Patents (2005) 15:1087-1103. In one embodiment, thespacer unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments,the phenylene portion of a p-amino benzyl unit is substituted with Qm,wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4. Examples of self-immolative spacerunits further include, but are not limited to, aromatic compounds thatare electronically similar to p-aminobenzyl alcohol (see, e.g., US2005/0256030 A1), such as 2 derivatives (Hay et al. (1999) Bioorg. Med.Chem. Lett. 9:2237) and ortho- or para-aminobenzylacetals. Spacers canbe used that undergo cyclization upon amide bond hydrolysis, such assubstituted and unsubstituted 4-aminobutyric acid amides (Rodrigues etal., Chemistry Biology, 1995, 2, 223); appropriately substitutedbicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J. Amer.Chem. Soc., 1972, 94, 5815); and 2 acid amides (Amsberry, et al., J.Org. Chem., 1990, 55, 5867). Elimination of amine-containing drugs thatare substituted at the a-position of glycine (Kingsbury, et al., J. Med.Chem., 1984, 27, 1447) are also examples of self-immolative spacersuseful in ADCs.

In one embodiment, a spacer unit is a branched bis(hydroxymethyl)styrene(BHMS) unit as depicted below, which can be used to incorporate andrelease multiple drugs.

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; n is 0 or 1; and p ranges raging from1 to about 20.

A linker may comprise any one or more of the above linker components. Incertain embodiments, a linker is as shown in brackets in the followingADC Formula II

Ab-([Aa-Ww-Yy]-D)_(p)  Formula II

wherein A is a stretcher unit, and a is an integer from 0 to 1; W is anamino acid unit, and w is an integer from 0 to 12; Y is a spacer unit,and y is 0, 1, or 2; and Ab, D, and p are defined as above for FormulaI. Exemplary embodiments of such linkers are described in US 20050238649A1, which is expressly incorporated herein by reference.

Exemplary linker components and combinations thereof are shown below inthe context of ADCs of Formula II:

Linkers components, including stretcher, spacer, and amino acid units,may be synthesized by methods known in the art, such as those describedin US 2005-0238649 A1.

Exemplary Drug Moieties

Maytansine and Maytansinoids

In some embodiments, an immunoconjugate comprises an antibody of theinvention conjugated to one or more maytansinoid molecules.Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody-drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification or derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through non-disulfide linkers to antibodies,(iii) stable in plasma, and (iv) effective against a variety of tumorcell lines.

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art and can be isolated from natural sources accordingto known methods or produced using genetic engineering techniques (seeYu et al (2002) PNAS 99:7968-7973). Maytansinol and maytansinolanalogues may also be prepared synthetically according to known methods.

Exemplary embodiments of maytansinoid drug moieties include: DM1; DM3;and DM4, as disclosed herein.

Auristatins and Dolastatins

In some embodiments, an immunoconjugate comprises an antibody of theinvention conjugated to dolastatin or a dolastatin peptidic analog orderivative, e.g., an auristatin (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in Senter et al,Proceedings of the American Association for Cancer Research, Volume 45,Abstract Number 623, presented Mar. 28, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

A peptidic drug moiety may be selected from Formulas D_(E) and D_(F)below:

wherein the wavy line of D_(E) and D_(F) indicates the covalentattachment site to an antibody or antibody-linker component, andindependently at each location:

R² is selected from H and C₁-C₈ alkyl;

R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from 2, 3, 4,5 and 6;

R⁶ is selected from H and C₁-C₈ alkyl;

R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈carbocycle and O—(C₁-C₈ alkyl);

R⁹ is selected from H and C₁-C₈ alkyl;

R¹⁰ selected from aryl or C₃-C₈ heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;

R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,—(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴ is H or C₁-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈ alkyl;

each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or—(CH₂)_(n)—COOH;

R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and

n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H or methyl. In an exemplary embodiment, R³ and R⁴are each isopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H. Instill another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹° is aryl.

In an exemplary embodiment, R¹° is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂).—SO₃H.

An exemplary auristatin embodiment of formula D_(E) is MMAE, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

An exemplary auristatin embodiment of formula D_(F) is MMAF, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate (see US 2005/0238649 and Doronina et al. (2006)Bioconjugate Chem. 17:114-124):

Other drug moieties include the following MMAF derivatives, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

In one aspect, hydrophilic groups including but not limited to,triethylene glycol esters (TEG), as shown above, can be attached to thedrug moiety at R¹¹. Without being bound by any particular theory, thehydrophilic groups assist in the internalization and non-agglomerationof the drug moiety.

Exemplary embodiments of ADCs of Formula I comprising anauristatin/dolastatin or derivative thereof are described in US2005-0238649 A1 and Doronina et al. (2006) Bioconjugate Chem.17:114-124, which is expressly incorporated herein by reference.Exemplary embodiments of ADCs of Formula I comprising MMAE or MMAF andvarious linker components have the following structures andabbreviations (wherein “Ab” is an antibody; p is 1 to about 8, “Val-Cit”is a valine-citrulline dipeptide; and “S” is a sulfur atom:

Exemplary embodiments of ADCs of Formula I comprising MMAF and variouslinker components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF.Interestingly, immunoconjugates comprising MMAF attached to an antibodyby a linker that is not proteolytically cleavable have been shown topossess activity comparable to immunoconjugates comprising MMAF attachedto an antibody by a proteolytically cleavable linker. See, Doronina etal. (2006) Bioconjugate Chem. 17:114-124. In such instances, drugrelease is believed to be effected by antibody degradation in the cell.Id.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. Auristatin/dolastatin drug moieties may beprepared according to the methods of: US 2005-0238649 A1; U.S. Pat. No.5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem.Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al(1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat.Biotechnol. 21(7):778-784.

In particular, auristatin/dolastatin drug moieties of formula D_(F),such as MMAF and derivatives thereof, may be prepared using methodsdescribed in US 2005-0238649 A1 and Doronina et al. (2006) BioconjugateChem. 17:114-124. Auristatin/dolastatin drug moieties of formula D_(E),such as MMAE and derivatives thereof, may be prepared using methodsdescribed in Doronina et al. (2003) Nat. Biotech. 21:778-784.Drug-linker moieties MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, andMC-vc-PAB-MMAE may be conveniently synthesized by routine methods, e.g.,as described in Doronina et al. (2003) Nat. Biotech. 21:778-784, andPatent Application Publication No. US 2005/0238649 A1, and thenconjugated to an antibody of interest.

Drug Loading

Drug loading is represented by p and is the average number of drugmoieties per antibody in a molecule of Formula I. Drug loading may rangefrom 1 to 20 drug moieties (D) per antibody. ADCs of Formula I includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See US 2005-0238649A1 (herein incorporated by reference in its entirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Only the most reactive lysine groups may react with an amine-reactivelinker reagent. Generally, antibodies do not contain many free andreactive cysteine thiol groups which may be linked to a drug moiety;indeed most cysteine thiol residues in antibodies exist as disulfidebridges. In certain embodiments, an antibody may be reduced with areducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine(TCEP), under partial or total reducing conditions, to generate reactivecysteine thiol groups. In certain embodiments, an antibody is subjectedto denaturing conditions to reveal reactive nucleophilic groups such aslysine or cysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachments (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography (see, e.g., Hamblett, K. J.,et al. “Effect of drug loading on the pharmacology, pharmacokinetics,and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No. 624,American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, S.C., et al. “Controlling the location of drug attachment in antibody-drugconjugates,” Abstract No. 627, American Association for Cancer Research,2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the AACR, Volume45, March 2004). In certain embodiments, a homogeneous ADC with a singleloading value may be isolated from the conjugation mixture byelectrophoresis or chromatography.

Certain Methods of Preparing Immunconjugates

An ADC of Formula I may be prepared by several routes employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) reaction of a nucleophilic group of an antibodywith a bivalent linker reagent to form Ab-L via a covalent bond,followed by reaction with a drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a bivalent linker reagent, toform D-L, via a covalent bond, followed by reaction with a nucleophilicgroup of an antibody. Exemplary methods for preparing an ADC of FormulaI via the latter route are described in US 20050238649 A1, which isexpressly incorporated herein by reference.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theantibody is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. Alternatively,sulfhydryl groups can be introduced into antibodies through modificationof lysine residues, e.g., by reacting lysine residues with 2 (Traut'sreagent), resulting in conversion of an amine into a thiol. Reactivethiol groups may be introduced into an antibody by introducing one, two,three, four, or more cysteine residues (e.g., by preparing variantantibodies comprising one or more non-native cysteine amino acidresidues).

Antibody-drug conjugates of the invention may also be produced byreaction between an electrophilic group on an antibody, such as analdehyde or ketone carbonyl group, with a nucleophilic group on a linkerreagent or drug. Useful nucleophilic groups on a linker reagent include,but are not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In oneembodiment, an antibody is modified to introduce electrophilic moietiesthat are capable of reacting with nucleophilic substituents on thelinker reagent or drug. In another embodiment, the sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the antibody that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, antibodies containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such analdehyde can be reacted with a drug moiety or linker nucleophile.

Nucleophilic groups on a drug moiety include, but are not limited to:amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide groups capable of reacting toform covalent bonds with electrophilic groups on linker moieties andlinker reagents including: (i) active esters such as NHS esters, HOBtesters, haloformates, and acid halides; (ii) alkyl and benzyl halidessuch as haloacetamides; (iii) aldehydes, ketones, carboxyl, andmaleimide groups.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with the following cross-linker reagents: BMPS,EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH,sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SLAB, sulfo-SMCC,and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) whichare commercially available (e.g., from Pierce Biotechnology, Inc.,Rockford, Ill., U.S.A; see pages 467-498, 2003-2004 ApplicationsHandbook and Catalog.

Immunoconjugates comprising an antibody and a cytotoxic agent may alsobe made using a variety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Alternatively, a fusion protein comprising an antibody and a cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.A recombinant DNA molecule may comprise regions encoding the antibodyand cytotoxic portions of the conjugate either adjacent to one anotheror separated by a region encoding a linker peptide which does notdestroy the desired properties of the conjugate.

In yet another embodiment, an antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pre-targeting whereinthe antibody-receptor conjugate is administered to the patient, followedby removal of unbound conjugate from the circulation using a clearingagent and then administration of a “ligand” (e.g., avidin) which isconjugated to a cytotoxic agent (e.g., a radionucleotide).

2. Exemplary Immunoconjugates—Thio Antibody Drug Conjugates

Preparation of Cysteine Engineered Anti-CD22 Antibodies

DNA encoding an amino acid sequence variant of the cysteine engineeredanti-CD22 antibodies and parent anti-CD22 antibodies of the invention isprepared by a variety of methods which include, but are not limited to,isolation from a natural source (in the case of naturally occurringamino acid sequence variants), preparation by site-directed (oroligonucleotide-mediated) mutagenesis (Carter (1985) et al Nucleic AcidsRes. 13:4431-4443; Ho et al (1989) Gene (Amst.) 77:51-59; Kunkel et al(1987) Proc. Natl. Acad. Sci. USA 82:488; Liu et al (1998) J. Biol.Chem. 273:20252-20260), PCR mutagenesis (Higuchi, (1990) in PCRProtocols, pp. 177-183, Academic Press; Ito et al (1991) Gene 102:67-70;Bernhard et al (1994) Bioconjugate Chem. 5:126-132; and Vallette et al(1989) Nuc. Acids Res. 17:723-733), and cassette mutagenesis (Wells etal (1985) Gene 34:315-323) of an earlier prepared DNA encoding thepolypeptide. Mutagenesis protocols, kits, and reagents are commerciallyavailable, e.g. QuikChange® Multi Site-Direct Mutagenesis Kit(Stratagene, La Jolla, Calif.). Single mutations are also generated byoligonucleotide directed mutagenesis using double stranded plasmid DNAas template by PCR based mutagenesis (Sambrook and Russel, (2001)Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et al (1983)Methods Enzymol. 100:468-500; Zoller, M. J. and Smith, M. (1982) Nucl.Acids Res. 10:6487-6500). Variants of recombinant antibodies may beconstructed also by restriction fragment manipulation or by overlapextension PCR with synthetic oligonucleotides. Mutagenic primers encodethe cysteine codon replacement(s). Standard mutagenesis techniques canbe employed to generate DNA encoding such mutant cysteine engineeredantibodies (Sambrook et al Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; andAusubel et al Current Protocols in Molecular Biology, Greene Publishingand Wiley-Interscience, New York, N.Y., 1993).

Phage display technology (McCafferty et al (1990) Nature 348:552-553)can be used to produce anti-CD22 human antibodies and antibody fragmentsin vitro, from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B-cell (Johnson et al (1993) Current Opinion inStructural Biology 3:564-571; Clackson et al (1991) Nature, 352:624-628;Marks et al (1991) J. Mol. Biol. 222:581-597; Griffith et al (1993) EMBOJ. 12:725-734; U.S. Pat. No. 5,565,332; U.S. Pat. No. 5,573,905; U.S.Pat. No. 5,567,610; U.S. Pat. No. 5,229,275).

Anti-CD22 antibodies may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. The appropriate amino acid sequence, or portionsthereof, may be produced by direct peptide synthesis using solid-phasetechniques (Stewart et al., Solid-Phase Peptide Synthesis, (1969) W.H.Freeman Co., San Francisco, Calif.; Merrifield, (1963) J. Am. Chem.Soc., 85:2149-2154). In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated solid phase synthesis maybe accomplished, for instance, employing t-BOC or Fmoc protected aminoacids and using an Applied Biosystems Peptide Synthesizer (Foster City,Calif.) using manufacturer's instructions. Various portions of theanti-CD22 antibody or CD22 polypeptide may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe desired anti-CD22 antibody or CD22 polypeptide.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (Morimoto et al (1992) Journal ofBiochemical and Biophysical Methods 24:107-117; and Brennan et al (1985)Science, 229:81), or produced directly by recombinant host cells. Fab,Fv and ScFv anti-CD22 antibody fragments can all be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of these fragments. Antibody fragments can be isolated from theantibody phage libraries discussed herein. Alternatively, Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab)₂ fragments (Carter et al (1992) Bio/Technology10:163-167), or isolated directly from recombinant host cell culture.The anti-CD22 antibody may be a (scFv) single chain Fv fragment (WO93/16185; U.S. Pat. No. 5,571,894; U.S. Pat. No. 5,587,458). Theanti-CD22 antibody fragment may also be a “linear antibody” (U.S. Pat.No. 5,641,870). Such linear antibody fragments may be monospecific orbispecific.

The description below relates primarily to production of anti-CD22antibodies by culturing cells transformed or transfected with a vectorcontaining anti-CD22 antibody-encoding nucleic acid. DNA encodinganti-CD22 antibodies may be obtained from a cDNA library prepared fromtissue believed to possess the anti-CD22 antibody mRNA and to express itat a detectable level. Accordingly, human anti-CD22 antibody or CD22polypeptide DNA can be conveniently obtained from a cDNA libraryprepared from human tissue. The anti-CD22 antibody-encoding gene mayalso be obtained from a genomic library or by known synthetic procedures(e.g., automated nucleic acid synthesis).

The design, selection, and preparation methods of the invention enablecysteine engineered anti-CD22 antibodies which are reactive withelectrophilic functionality. These methods further enable antibodyconjugate compounds such as antibody-drug conjugate (ADC) compounds withdrug molecules at designated, designed, selective sites. Reactivecysteine residues on an antibody surface allow specifically conjugatinga drug moiety through a thiol reactive group such as maleimide orhaloacetyl. The nucleophilic reactivity of the thiol functionality of aCys residue to a maleimide group is about 1000 times higher compared toany other amino acid functionality in a protein, such as amino group oflysine residues or the N-terminal amino group. Thiol specificfunctionality in iodoacetyl and maleimide reagents may react with aminegroups, but higher pH (>9.0) and longer reaction times are required(Garman, 1997, Non-Radioactive Labelling: A Practical Approach, AcademicPress, London). The amount of free thiol in a protein may be estimatedby the standard Ellman's assay. Immunoglobulin M is an example of adisulfide-linked pentamer, while immunoglobulin G is an example of aprotein with internal disulfide bridges bonding the subunits together.In proteins such as this, reduction of the disulfide bonds with areagent such as dithiothreitol (DTT) or selenol (Singh et al (2002)Anal. Biochem. 304:147-156) is required to generate the reactive freethiol. This approach may result in loss of antibody tertiary structureand antigen binding specificity.

The Pheselector (Phage ELISA for Selection of Reactive Thiols) Assayallows for detection of reactive cysteine groups in antibodies in anELISA phage format thereby assisting in the design of cysteineengineered antibodies (WO 2006/034488). The cysteine engineered antibodyis coated on well surfaces, followed by incubation with phage particles,addition of HRP labeled secondary antibody, and absorbance detection.Mutant proteins displayed on phage may be screened in a rapid, robust,and high-throughput manner. Libraries of cysteine engineered antibodiescan be produced and subjected to binding selection using the sameapproach to identify appropriately reactive sites of free Cysincorporation from random protein-phage libraries of antibodies or otherproteins. This technique includes reacting cysteine mutant proteinsdisplayed on phage with an affinity reagent or reporter group which isalso thiol-reactive.

The PHESELECTOR assay allows screening of reactive thiol groups inantibodies. Identification of the A121C variant by this method isexemplary. The entire Fab molecule may be effectively searched toidentify more ThioFab variants with reactive thiol groups. A parameter,fractional surface accessibility, was employed to identify andquantitate the accessibility of solvent to the amino acid residues in apolypeptide. The surface accessibility can be expressed as the surfacearea (Å²) that can be contacted by a solvent molecule, e.g. water. Theoccupied space of water is approximated as a 1.4 Å radius sphere.Software is freely available or licensable (Secretary to CCP4, DaresburyLaboratory, Warrington, Wash.4 4AD, United Kingdom, Fax: (+44) 1925603825, or by internet: www.ccp4.ac.uk/dist/html/INDEX.html) as the CCP4Suite of crystallography programs which employ algorithms to calculatethe surface accessibility of each amino acid of a protein with knownx-ray crystallography derived coordinates (“The CCP4 Suite: Programs forProtein Crystallography” (1994) Acta. Cryst. D50:760-763). Two exemplarysoftware modules that perform surface accessibility calculations are“AREAIMOL” and “SURFACE”, based on the algorithms of B. Lee and F. M.Richards (1971) J. Mol. Biol. 55:379-400. AREAIMOL defines the solventaccessible surface of a protein as the locus of the centre of a probesphere (representing a solvent molecule) as it rolls over the Van derWaals surface of the protein. AREAIMOL calculates the solvent accessiblesurface area by generating surface points on an extended sphere abouteach atom (at a distance from the atom centre equal to the sum of theatom and probe radii), and eliminating those that lie within equivalentspheres associated with neighboring atoms. AREAIMOL finds the solventaccessible area of atoms in a PDB coordinate file, and summarizes theaccessible area by residue, by chain and for the whole molecule.Accessible areas (or area differences) for individual atoms can bewritten to a pseudo-PDB output file. AREAIMOL assumes a single radiusfor each element, and only recognizes a limited number of differentelements.

AREAIMOL and SURFACE report absolute accessibilities, i.e. the number ofsquare Angstroms (Å). Fractional surface accessibility is calculated byreference to a standard state relevant for an amino acid within apolypeptide. The reference state is tripeptide Gly-X-Gly, where X is theamino acid of interest, and the reference state should be an ‘extended’conformation, i.e. like those in beta-strands. The extended conformationmaximizes the accessibility of X. A calculated accessible area isdivided by the accessible area in a Gly-X-Gly tripeptide reference stateand reports the quotient, which is the fractional accessibility. Percentaccessibility is fractional accessibility multiplied by 100. Anotherexemplary algorithm for calculating surface accessibility is based onthe SOLV module of the program xsae (Broger, C., F. Hoffman-LaRoche,Basel) which calculates fractional accessibility of an amino acidresidue to a water sphere based on the X-ray coordinates of thepolypeptide. The fractional surface accessibility for every amino acidin an antibody may be calculated using available crystal structureinformation (Eigenbrot et al. (1993) J Mol. Biol. 229:969-995).

DNA encoding the cysteine engineered antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, orother mammalian host cells, such as myeloma cells (U.S. Pat. No.5,807,715; US 2005/0048572; US 2004/0229310) that do not otherwiseproduce the antibody protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells.

After design and selection, cysteine engineered antibodies, e.g.ThioFabs, with the engineered, highly reactive unpaired Cys residues,may be produced by: (i) expression in a bacterial, e.g. E. coli, system(Skerra et al (1993) Curr. Opinion in Immunol. 5:256-262; Pliickthun(1992) Immunol. Revs. 130:151-188) or a mammalian cell culture system(WO 01/00245), e.g. Chinese Hamster Ovary cells (CHO); and (ii)purification using common protein purification techniques (Lowman et al(1991) J. Biol. Chem. 266(17):10982-10988).

The engineered Cys thiol groups react with electrophilic linker reagentsand drug-linker intermediates to form cysteine engineered antibody drugconjugates and other labelled cysteine engineered antibodies. Cysresidues of cysteine engineered antibodies, and present in the parentantibodies, which are paired and form interchain and intrachaindisulfide bonds do not have any reactive thiol groups (unless treatedwith a reducing agent) and do not react with electrophilic linkerreagents or drug-linker intermediates. The newly engineered Cys residue,can remain unpaired, and able to react with, i.e. conjugate to, anelectrophilic linker reagent or drug-linker intermediate, such as adrug-maleimide. Exemplary drug-linker intermediates include: MC-MMAE,MC-MMAF, MC-vc-PAB-MMAE, and MC-vc-PAB-MMAF. The structure positions ofthe engineered Cys residues of the heavy and light chains are numberedaccording to a sequential numbering system. This sequential numberingsystem is correlated to the Kabat numbering system (Kabat et al., (1991)Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md.) starting at theN-terminus, differs from the Kabat numbering scheme (bottom row) byinsertions noted by a,b,c. Using the Kabat numbering system, the actuallinear amino acid sequence may contain fewer or additional amino acidscorresponding to a shortening of, or insertion into, a FR or CDR of thevariable domain. The cysteine engineered heavy chain variant sites areidentified by the sequential numbering and Kabat numbering schemes.

In one embodiment, the cysteine engineered anti-CD22 antibody isprepared by a process comprising:

(a) replacing one or more amino acid residues of a parent anti-CD22antibody by cysteine; and

(b) determining the thiol reactivity of the cysteine engineeredanti-CD22 antibody by reacting the cysteine engineered antibody with athiol-reactive reagent.

The cysteine engineered antibody may be more reactive than the parentantibody with the thiol-reactive reagent.

The free cysteine amino acid residues may be located in the heavy orlight chains, or in the constant or variable domains. Antibodyfragments, e.g. Fab, may also be engineered with one or more cysteineamino acids replacing amino acids of the antibody fragment, to formcysteine engineered antibody fragments.

Another embodiment of the invention provides a method of preparing(making) a cysteine engineered anti-CD22 antibody, comprising:

(a) introducing one or more cysteine amino acids into a parent anti-CD22antibody in order to generate the cysteine engineered anti-CD22antibody; and

(b) determining the thiol reactivity of the cysteine engineered antibodywith a thiol-reactive reagent;

wherein the cysteine engineered antibody is more reactive than theparent antibody with the thiol-reactive reagent.

Step (a) of the method of preparing a cysteine engineered antibody maycomprise:

(i) mutagenizing a nucleic acid sequence encoding the cysteineengineered antibody;

(ii) expressing the cysteine engineered antibody; and

(iii) isolating and purifying the cysteine engineered antibody.

Step (b) of the method of preparing a cysteine engineered antibody maycomprise expressing the cysteine engineered antibody on a viral particleselected from a phage or a phagemid particle.

Step (b) of the method of preparing a cysteine engineered antibody mayalso comprise:

(i) reacting the cysteine engineered antibody with a thiol-reactiveaffinity reagent to generate an affinity labelled, cysteine engineeredantibody; and

(ii) measuring the binding of the affinity labelled, cysteine engineeredantibody to a capture media.

Another embodiment of the invention is a method of screening cysteineengineered antibodies with highly reactive, unpaired cysteine aminoacids for thiol reactivity comprising:

(a) introducing one or more cysteine amino acids into a parent antibodyin order to generate a cysteine engineered antibody;

(b) reacting the cysteine engineered antibody with a thiol-reactiveaffinity reagent to generate an affinity labelled, cysteine engineeredantibody; and

(c) measuring the binding of the affinity labelled, cysteine engineeredantibody to a capture media; and

(d) determining the thiol reactivity of the cysteine engineered antibodywith the thiol-reactive reagent.

Step (a) of the method of screening cysteine engineered antibodies maycomprise:

(i) mutagenizing a nucleic acid sequence encoding the cysteineengineered antibody;

(ii) expressing the cysteine engineered antibody; and

(iii) isolating and purifying the cysteine engineered antibody.

Step (b) of the method of screening cysteine engineered antibodies maycomprise expressing the cysteine engineered antibody on a viral particleselected from a phage or a phagemid particle.

Step (b) of the method of screening cysteine engineered antibodies mayalso comprise:

(i) reacting the cysteine engineered antibody with a thiol-reactiveaffinity reagent to generate an affinity labelled, cysteine engineeredantibody; and

(ii) measuring the binding of the affinity labelled, cysteine engineeredantibody to a capture media.

Cysteine Engineering of Anti-CD22 10F4 IgG Variants

Cysteine was introduced at the heavy chain 118 (EU numbering)(equivalent to heavy chain position 121, sequential numbering) site intothe full-length, chimeric parent monoclonal anti-CD22 antibodies by thecysteine engineering methods described herein.

The parent antibody, “std Anti-CD22 Hu 10F4v3 Fc” (Heavy Chain sequence:SEQ ID NO:88, Light Chain sequence: SEQ ID NO:87, FIG. 5B) was cysteineengineered to give “A118C thio hu anti-CD22 10F4v3” (Heavy Chainsequence: SEQ ID NO:92, Light Chain sequence: SEQ ID NO:91, FIG. 17),“S400C thio hu anti-CD22 10F4v3” (Heavy Chain sequence: SEQ ID NO:93,Light Chain sequence: SEQ ID NO:91, FIG. 17), or “V205C thio anti-CD2210F4v3” (Heavy Chain sequence: SEQ ID NO:88, Light Chain sequence: SEQID NO:91, FIGS. 5B and 17).

These cysteine engineered monoclonal antibodies were expressed in CHO(Chinese Hamster Ovary) cells by transient fermentation in mediacontaining 1 mM cysteine.

Labelled Cysteine Engineered Anti-CD22 Antibodies

Cysteine engineered anti-CD22 antibodies may be site-specifically andefficiently coupled with a thiol-reactive reagent. The thiol-reactivereagent may be a multifunctional linker reagent, a capture, i.e.affinity, label reagent (e.g. a biotin-linker reagent), a detectionlabel (e.g. a fluorophore reagent), a solid phase immobilization reagent(e.g. SEPHAROSE™, polystyrene, or glass), or a drug-linker intermediate.One example of a thiol-reactive reagent is N-ethyl maleimide (NEM). Inan exemplary embodiment, reaction of a ThioFab with a biotin-linkerreagent provides a biotinylated ThioFab by which the presence andreactivity of the engineered cysteine residue may be detected andmeasured. Reaction of a ThioFab with a multifunctional linker reagentprovides a ThioFab with a functionalized linker which may be furtherreacted with a drug moiety reagent or other label. Reaction of a ThioFabwith a drug-linker intermediate provides a ThioFab drug conjugate.

The exemplary methods described here may be applied generally to theidentification and production of antibodies, and more generally, toother proteins through application of the design and screening stepsdescribed herein.

Such an approach may be applied to the conjugation of otherthiol-reactive reagents in which the reactive group is, for example, amaleimide, an iodoacetamide, a pyridyl disulfide, or otherthiol-reactive conjugation partner (Haugland, 2003, Molecular ProbesHandbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997,Non-Radioactive Labelling: A Practical Approach, Academic Press, London;Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in BioconjugateTechniques (1996) Academic Press, San Diego, pp. 40-55, 643-671). Thethiol-reactive reagent may be a drug moiety, a fluorophore such as afluorescent dye like fluorescein or rhodamine, a chelating agent for animaging or radiotherapeutic metal, a peptidyl or non-peptidyl label ordetection tag, or a clearance-modifying agent such as various isomers ofpolyethylene glycol, a peptide that binds to a third component, oranother carbohydrate or lipophilic agent.

Uses of Cysteine Engineered Anti-CD22 Antibodies

Cysteine engineered anti-CD22 antibodies, and conjugates thereof mayfind use as therapeutic and/or diagnostic agents. The present inventionfurther provides methods of preventing, managing, treating orameliorating one or more symptoms associated with a B-cell relateddisorder. In particular, the present invention provides methods ofpreventing, managing, treating, or ameliorating one or more symptomsassociated with a cell proliferative disorder, such as cancer, e.g.,lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsedaggressive NHL, relapsed indolent NHL, refractory NHL, refractoryindolent NHL, chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocyticleukemia (ALL), and mantle cell lymphoma. The present invention stillfurther provides methods for diagnosing a CD22 related disorder orpredisposition to developing such a disorder, as well as methods foridentifying antibodies, and antigen-binding fragments of antibodies,that preferentially bind B cell-associated CD22 polypeptides.

Another embodiment of the present invention is directed to the use of acysteine engineered anti-CD22 antibody for the preparation of amedicament useful in the treatment of a condition which is responsive toa B cell related disorder.

Cysteine Engineered Antibody Drug Conjugates (Thio-Antibody DrugConjugates)

Another aspect of the invention is an antibody-drug conjugate compoundcomprising a cysteine engineered anti-CD22 antibody (Ab), and anauristatin drug moiety (D) wherein the cysteine engineered antibody isattached through one or more free cysteine amino acids by a linkermoiety (L) to D; the compound having Formula I:

Ab-(L-D)_(p)  I

where p is 1, 2, 3, or 4; and wherein the cysteine engineered antibodyis prepared by a process comprising replacing one or more amino acidresidues of a parent anti-CD22 antibody by one or more free cysteineamino acids.

FIG. 10 shows embodiments of cysteine engineered anti-CD22 antibody drugconjugates (ADC) where an auristatin drug moiety is attached to anengineered cysteine group in: the light chain (LC-ADC); the heavy chain(HC-ADC); and the Fc region (Fc-ADC).

Potential advantages of cysteine engineered anti-CD22 antibody drugconjugates include improved safety (larger therapeutic index), improvedPK parameters, the antibody inter-chain disulfide bonds are retainedwhich may stabilize the conjugate and retain its active bindingconformation, the sites of drug conjugation are defined, and thepreparation of cysteine engineered antibody drug conjugates fromconjugation of cysteine engineered antibodies to drug-linker reagentsresults in a more homogeneous product.

Linkers

“Linker”, “Linker Unit”, or “link” means a chemical moiety comprising acovalent bond or a chain of atoms that covalently attaches an antibodyto a drug moiety. In various embodiments, a linker is specified as L. A“Linker” (L) is a bifunctional or multifunctional moiety which can beused to link one or more Drug moieties (D) and an antibody unit (Ab) toform antibody-drug conjugates (ADC) of Formula I. Antibody-drugconjugates (ADC) can be conveniently prepared using a Linker havingreactive functionality for binding to the Drug and to the Antibody. Acysteine thiol of a cysteine engineered antibody (Ab) can form a bondwith an electrophilic functional group of a linker reagent, a drugmoiety or drug-linker intermediate.

In one aspect, a Linker has a reactive site which has an electrophilicgroup that is reactive to a nucleophilic cysteine present on anantibody. The cysteine thiol of the antibody is reactive with anelectrophilic group on a Linker and forms a covalent bond to a Linker.Useful electrophilic groups include, but are not limited to, maleimideand haloacetamide groups.

Linkers include a divalent radical such as an alkyldiyl, an arylene, aheteroarylene, moieties such as: —(CR₂)_(n)O(CR₂)_(n)—, repeating unitsof alkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino(e.g. polyethyleneamino, Jeffamine™); and diacid ester and amidesincluding succinate, succinamide, diglycolate, malonate, and caproamide.

Cysteine engineered antibodies react with linker reagents or drug-linkerintermediates, with electrophilic functional groups such as maleimide orα-halo carbonyl, according to the conjugation method at page 766 ofKlussman, et al (2004), Bioconjugate Chemistry 15(4):765-773, andaccording to the protocol of Example x.

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe” or “af”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl4-(2-pyridylthio)pentanoate (“SPP”), N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), N-Succinimidyl(4-iodo-acetyl) aminobenzoate (“STAB”), ethyleneoxy —CH₂CH₂O— as one ormore repeating units (“EO” or “PEO”). Additional linker components areknown in the art and some are described herein.

In one embodiment, linker L of an ADC has the formula:

A_(a)-W_(w)-Y_(y)-

wherein:

-A- is a Stretcher unit covalently attached to a cysteine thiol of theantibody (Ab);

a is 0 or 1;

each -W- is independently an Amino Acid unit;

w is independently an integer ranging from 0 to 12;

-Y- is a Spacer unit covalently attached to the drug moiety; and

y is 0, 1 or 2.

Stretcher Unit

The Stretcher unit (-A-), when present, is capable of linking anantibody unit to an amino acid unit (-W-). In this regard an antibody(Ab) has a functional group that can form a bond with a functional groupof a Stretcher. Useful functional groups that can be present on anantibody, either naturally or via chemical manipulation include, but arenot limited to, sulfhydryl (—SH), amino, hydroxyl, carboxy, the anomerichydroxyl group of a carbohydrate, and carboxyl. In one aspect, theantibody functional groups are sulfhydryl or amino. Sulfhydryl groupscan be generated by reduction of an intramolecular disulfide bond of anantibody. Alternatively, sulfhydryl groups can be generated by reactionof an amino group of a lysine moiety of an antibody using2-iminothiolane (Traut's reagent) or another sulfhydryl generatingreagent. In one embodiment, an antibody (Ab) has a free cysteine thiolgroup that can form a bond with an electrophilic functional group of aStretcher Unit. Exemplary stretcher units in Formula I conjugates aredepicted by Formulas II and III, wherein Ab-, -W-, -Y-, -D, w and y areas defined above, and R¹⁷ is a divalent radical selected from (CH₂)_(r),C₃-C₈ carbocyclyl, O—(CH₂)_(r), arylene, (CH₂)_(r)-arylene,-arylene-(CH₂)_(r)—, (CH₂)_(r)—(C₃-C₈ carbocyclyl), (C₃-C₈carbocyclyl)-(CH₂)_(r), C₃-C₈ heterocyclyl, (CH₂)_(r)—(C₃-C₈heterocyclyl), —(C₃-C₈ heterocyclyl)-(CH₂)_(r)—,—(CH₂)_(r)C(O)NR^(b)(CH₂)_(r)—, —(CH₂CH₂O)_(r)—, —(CH₂CH₂O)_(r)—CH₂—,—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—,—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂—,—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂CH₂O)C,—(CH₂CH₂O)_(n)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂—, and—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂)_(r)—; where R^(b) is H, C₁-C₆ alkyl,phenyl, or benzyl; and r is independently an integer ranging from 1-10.

Arylene includes divalent aromatic hydrocarbon radicals of 6-20 carbonatoms derived by the removal of two hydrogen atoms from the aromaticring system. Typical arylene groups include, but are not limited to,radicals derived from benzene, substituted benzene, naphthalene,anthracene, biphenyl, and the like.

Heterocyclyl groups include a ring system in which one or more ringatoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 1 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo[4,5], [5,5], [5,6], or [6,6]system. Heterocycles are described in Paquette, Leo A.; “Principles ofModern Heterocyclic Chemistry” (W.A. Benjamin, New York, 1968),particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4Ah-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

Carbocyclyl groups include a saturated or unsaturated ring having 3 to 7carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle.Monocyclic carbocycles have 3 to 6 ring atoms, still more typically 5 or6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g.arranged as a bicyclo[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10ring atoms arranged as a bicyclo[5,6] or [6,6] system. Examples ofmonocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

It is to be understood from all the exemplary embodiments of Formula IADC such as II-VI, that even where not denoted expressly, from 1 to 4drug moieties are linked to an antibody (p=1-4), depending on the numberof engineered cysteine residues.

An illustrative Formula II Stretcher unit is derived frommaleimido-caproyl (MC) wherein R¹⁷ is —(CH₂)₅—:

An illustrative Stretcher unit of Formula II, and is derived frommaleimidopropanoyl (MP) wherein R¹⁷ is —(CH₂)₂—:

Another illustrative Stretcher unit of Formula II wherein R¹⁷ is—(CH₂CH₂O)_(r)—CH₂— and r is 2:

Another illustrative Stretcher unit of Formula II wherein R¹⁷ is—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂— where R^(b) is H and each r is 2:

An illustrative Stretcher unit of Formula III wherein R¹⁷ is —(CH₂)₅—:

In another embodiment, the Stretcher unit is linked to the cysteineengineered anti-CD22 antibody via a disulfide bond between theengineered cystein sulfur atom of the antibody and a sulfur atom of theStretcher unit. A representative Stretcher unit of this embodiment isdepicted by Formula IV, wherein R¹⁷, Ab-, -W-, -Y-, -D, w and y are asdefined above.

Ab-SS—R¹⁷—C(O)-W_(w)Y_(y)-D)_(p)  IV

In yet another embodiment, the reactive group of the Stretcher containsa thiol-reactive functional group that can form a bond with a freecysteine thiol of an antibody. Examples of thiol-reaction functionalgroups include, but are not limited to, maleimide, α-haloacetyl,activated esters such as succinimide esters, 4-nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acidchlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative Stretcher units of this embodiment are depicted byFormulas Va and Vb, wherein —R¹⁷—, Ab-, -W-, -Y-, -D, w and y are asdefined above;

Ab-SC(O)NH—R¹⁷—C(O)-W_(w)-Y_(y)-D)_(p)  Va

Ab-SC(S)NH—R¹⁷—C(O)-W_(w)-Y_(y)-D)_(p)  Vb

In another embodiment, the linker may be a dendritic type linker forcovalent attachment of more than one drug moiety through a branching,multifunctional linker moiety to an antibody (Sun et al (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)Bioorganic & Medicinal Chemistry 11:1761-1768; King (2002) TetrahedronLetters 43:1987-1990). Dendritic linkers can increase the molar ratio ofdrug to antibody, i.e. loading, which is related to the potency of theADC. Thus, where a cysteine engineered antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Amino Acid Unit

The linker may comprise amino acid residues. The Amino Acid unit(-W_(w)-), when present, links the antibody (Ab) to the drug moiety (D)of the cysteine engineered antibody-drug conjugate (ADC) of theinvention.

-W_(w)- is a dipeptide, tripeptide, tetrapeptide, pentapeptide,hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,undecapeptide or dodecapeptide unit. Amino acid residues which comprisethe Amino Acid unit include those occurring naturally, as well as minoramino acids and non-naturally occurring amino acid analogs, such ascitrulline. Each -W- unit independently has the formula denoted below inthe square brackets, and w is an integer ranging from 0 to 12:

wherein R¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

When R¹⁹ is other than hydrogen, the carbon atom to which R¹⁹ isattached is chiral. Each carbon atom to which R¹⁹ is attached isindependently in the (S) or (R) configuration, or a racemic mixture.Amino acid units may thus be enantiomerically pure, racemic, ordiastereomeric.

Exemplary -W_(w)- Amino Acid units include a dipeptide, a tripeptide, atetrapeptide or a pentapeptide. Exemplary dipeptides include:valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline.

The Amino Acid unit can be enzymatically cleaved by one or more enzymes,including a tumor-associated protease, to liberate the Drug moiety (-D),which in one embodiment is protonated in vivo upon release to provide aDrug (D). Amino acid linker components can be designed and optimized intheir selectivity for enzymatic cleavage by a particular enzymes, forexample, a tumor-associated protease, cathepsin B, C and D, or a plasminprotease.

Spacer Unit

The Spacer unit (-Y_(y)-), when present (y=1 or 2), links an Amino Acidunit (-W_(w)-) to the drug moiety (D) when an Amino Acid unit is present(w=1-12). Alternately, the Spacer unit links the Stretcher unit to theDrug moiety when the Amino Acid unit is absent. The Spacer unit alsolinks the drug moiety to the antibody unit when both the Amino Acid unitand Stretcher unit are absent (w, y=0). Spacer units are of two generaltypes: self-immolative and non self-immolative. A non self-immolativeSpacer unit is one in which part or all of the Spacer unit remains boundto the Drug moiety after cleavage, particularly enzymatic, of an AminoAcid unit from the antibody-drug conjugate or the Drug moiety-linker.When an ADC containing a glycine-glycine Spacer unit or a glycine Spacerunit undergoes enzymatic cleavage via a tumor-cell associated-protease,a cancer-cell-associated protease or a lymphocyte-associated protease, aglycine-glycine-Drug moiety or a glycine-Drug moiety is cleaved fromAb-A_(a)-Ww-. In one embodiment, an independent hydrolysis reactiontakes place within the target cell, cleaving the glycine-Drug moietybond and liberating the Drug.

In another embodiment, -Y_(y)- is a p-aminobenzylcarbamoyl (PAB) unitwhose phenylene portion is substituted with Q_(m) wherein Q is —C₁-C₈alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano; and m is an integerranging from 0-4.

Exemplary embodiments of a non self-immolative Spacer unit (-Y-) are:-Gly-Gly-; -Gly-; -Ala-Phe-; -Val-Cit-.

In one embodiment, a Drug moiety-linker or an ADC is provided in whichthe Spacer unit is absent (y=0), or a pharmaceutically acceptable saltor solvate thereof.

Alternatively, an ADC containing a self-immolative Spacer unit canrelease -D. In one embodiment, -Y- is a PAB group that is linked to-W_(w)- via the amino nitrogen atom of the PAB group, and connecteddirectly to -D via a carbonate, carbamate or ether group, where the ADChas the exemplary structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; and p ranges from 1 to 4.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999)Bioorg. Med. Chem. Lett. 9:2237), heterocyclic PAB analogs (US2005/0256030), beta-glucuronide (WO 2007/011968), and ortho orpara-aminobenzylacetals. Spacers can be used that undergo cyclizationupon amide bond hydrolysis, such as substituted and unsubstituted4-aminobutyric acid amides (Rodrigues et al (1995) Chemistry Biology2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ringsystems (Storm et al (1972) J. Amer. Chem. Soc. 94:5815) and 2 acidamides (Amsberry, et al (1990) J. Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at glycine (Kingsbury et al(1984) J. Med. Chem. 27:1447) are also examples of self-immolativespacer useful in ADCs.

Exemplary Spacer units (-Y_(y)-) are represented by Formulas X-XII:

Dendritic Linkers

In another embodiment, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety through a branching,multifunctional linker moiety to an antibody (Sun et al (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers canincrease the molar ratio of drug to antibody, i.e. loading, which isrelated to the potency of the ADC. Thus, where a cysteine engineeredantibody bears only one reactive cysteine thiol group, a multitude ofdrug moieties may be attached through a dendritic linker. Exemplaryembodiments of branched, dendritic linkers include2,6-bis(hydroxymethyl)-p-cresol and 2,4,6-tris(hydroxymethyl)-phenoldendrimer units (WO 2004/01993; Szalai et al (2003) J. Amer. Chem. Soc.125:15688-15689; Shamis et al (2004) J. Amer. Chem. Soc. 126:1726-1731;Amir et al (2003) Angew. Chem. Int. Ed. 42:4494-4499).

In one embodiment, the Spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS), which can be used to incorporate andrelease multiple drugs, having the structure:

comprising a 2-(4-aminobenzylidene)propane-1,3-diol dendrimer unit (WO2004/043493; de Groot et al (2003) Angew. Chem. Int. Ed. 42:4490-4494),wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; n is 0 or 1; and p ranges ranging from1 to 4.

Exemplary embodiments of the Formula I antibody-drug conjugate compoundsinclude XIIIa (MC), XIIIb (val-cit), XIIIc (MC-val-cit), and XIIId(MC-val-cit-PAB):

Other exemplary embodiments of the Formula Ia antibody-drug conjugatecompounds include XIVa-e:

where X is:

Y is:

and R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

In another embodiment, a Linker has a reactive functional group whichhas a nucleophilic group that is reactive to an electrophilic grouppresent on an antibody. Useful electrophilic groups on an antibodyinclude, but are not limited to, aldehyde and ketone carbonyl groups.The heteroatom of a nucleophilic group of a Linker can react with anelectrophilic group on an antibody and form a covalent bond to anantibody unit. Useful nucleophilic groups on a Linker include, but arenot limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide. The electrophilic group on anantibody provides a convenient site for attachment to a Linker.

Typically, peptide-type Linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to the liquidphase synthesis method (E. Schroder and K. Liibke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press) which is well known in the field ofpeptide chemistry. Linker intermediates may be assembled with anycombination or sequence of reactions including Spacer, Stretcher, andAmino Acid units. The Spacer, Stretcher, and Amino Acid units may employreactive functional groups which are electrophilic, nucleophilic, orfree radical in nature. Reactive functional groups include, but are notlimited to carboxyls, hydroxyls, para-nitrophenylcarbonate,isothiocyanate, and leaving groups, such as O-mesyl, O-tosyl, —Cl, —Br,—I; or maleimide.

In another embodiment, the Linker may be substituted with groups whichmodulated solubility or reactivity. For example, a charged substituentsuch as sulfonate (—SO₃ ⁻) or ammonium, may increase water solubility ofthe reagent and facilitate the coupling reaction of the linker reagentwith the antibody or the drug moiety, or facilitate the couplingreaction of Ab-L (antibody-linker intermediate) with D, or D-L(drug-linker intermediate) with Ab, depending on the synthetic routeemployed to prepare the ADC.

Linker Reagents

Conjugates of the antibody and auristatin may be made using a variety ofbifunctional linker reagents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6 and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

The antibody drug conjugates may also be prepared with linker reagents:BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB,SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-STAB,sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate), and including bis-maleimidereagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO)₃, and BM(PEO)₄, which arecommercially available from Pierce Biotechnology, Inc., Customer ServiceDepartment, P.O. Box 117, Rockford, Ill. 61105 U.S.A, U.S.A1-800-874-3723, International +815-968-0747. Bis-maleimide reagentsallow the attachment of the thiol group of a cysteine engineeredantibody to a thiol-containing drug moiety, label, or linkerintermediate, in a sequential or concurrent fashion. Other functionalgroups besides maleimide, which are reactive with a thiol group of acysteine engineered antibody, drug moiety, label, or linker intermediateinclude iodoacetamide, bromoacetamide, vinyl pyridine, disulfide,pyridyl disulfide, isocyanate, and isothiocyanate.

Useful linker reagents can also be obtained via other commercialsources, such as Molecular Biosciences Inc. (Boulder, Colo.), orsynthesized in accordance with procedures described in Toki et al (2002)J. Org. Chem. 67:1866-1872; Walker, M. A. (1995) J. Org. Chem.60:5352-5355; Frisch et al (1996) Bioconjugate Chem. 7:180-186; U.S.Pat. No. 6,214,345; WO 02/088172; US 2003130189; US2003096743; WO03/026577; WO 03/043583; and WO 04/032828.

Stretchers of formula (IIIa) can be introduced into a Linker by reactingthe following linker reagents with the N-terminus of an Amino Acid unit:

where n is an integer ranging from 1-10 and T is —H or —SO₃Na;

where n is an integer ranging from 0-3;

Stretcher units of can be introduced into a Linker by reacting thefollowing bifunctional reagents with the N-terminus of an Amino Acidunit:

where X is Br or I.

Stretcher units of formula can also be introduced into a Linker byreacting the following bifunctional reagents with the N-terminus of anAmino Acid unit:

An exemplary valine-citrulline (val-cit or vc) dipeptide linker reagenthaving a maleimide Stretcher and a para-aminobenzylcarbamoyl (PAB)self-immolative Spacer has the structure:

An exemplary phe-lys(Mtr, mono-4-methoxytrityl) dipeptide linker reagenthaving a maleimide Stretcher unit and a PAB self-immolative Spacer unitcan be prepared according to Dubowchik, et al. (1997) TetrahedronLetters, 38:5257-60, and has the structure:

Exemplary antibody-drug conjugate compounds of the invention include:

where Val is valine; Cit is citrulline; p is 1, 2, 3, or 4; and Ab is acysteine engineered anti-CD22 antibody.

Preparation of Cysteine Engineered Anti-CD22 Antibody-Drug Conjugates

The ADC of Formula I may be prepared by several routes, employingorganic chemistry reactions, conditions, and reagents known to thoseskilled in the art, including: (1) reaction of a cysteine group of acysteine engineered antibody with a linker reagent, to formantibody-linker intermediate Ab-L, via a covalent bond, followed byreaction with an activated drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a linker reagent, to formdrug-linker intermediate D-L, via a covalent bond, followed by reactionwith a cysteine group of a cysteine engineered antibody. Conjugationmethods (1) and (2) may be employed with a variety of cysteineengineered antibodies, drug moieties, and linkers to prepare theantibody-drug conjugates of Formula I.

Antibody cysteine thiol groups are nucleophilic and capable of reactingto form covalent bonds with electrophilic groups on linker reagents anddrug-linker intermediates including: (i) active esters such as NHSesters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides, such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups; and (iv) disulfides, including pyridyldisulfides, via sulfide exchange. Nucleophilic groups on a drug moietyinclude, but are not limited to: amine, thiol, hydroxyl, hydrazide,oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide groups capable of reacting to form covalent bonds withelectrophilic groups on linker moieties and linker reagents.

Cysteine engineered antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(Cleland's reagent, dithiothreitol) or TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.), followed byreoxidation to reform interchain and intrachain disulfide bonds (Examplex). For example, full length, cysteine engineered monoclonal antibodies(ThioMabs) expressed in CHO cells are reduced with about a 50 foldexcess of TCEP for 3 hrs at 37° C. to reduce disulfide bonds in cysteineadducts which may form between the newly introduced cysteine residuesand the cysteine present in the culture media. The reduced ThioMab isdiluted and loaded onto HiTrap S column in 10 mM sodium acetate, pH 5,and eluted with PBS containing 0.3M sodium chloride. Disulfide bondswere reestablished between cysteine residues present in the parent Mabwith dilute (200 nM) aqueous copper sulfate (CuSO₄) at room temperature,overnight. Alternatively, dehydroascorbic acid (DHAA) is an effectiveoxidant to reestablish the intrachain disulfide groups of the cysteineengineered antibody after reductive cleavage of the cysteine adducts.Other oxidants, i.e. oxidizing agents, and oxidizing conditions, whichare known in the art may be used. Ambient air oxidation is alsoeffective. This mild, partial reoxidation step forms intrachaindisulfides efficiently with high fidelity and preserves the thiol groupsof the newly introduced cysteine residues. An approximate 10 fold excessof drug-linker intermediate, e.g. MC-vc-PAB-MMAE, was added, mixed, andlet stand for about an hour at room temperature to effect conjugationand form the 10F4v3 anti-CD22 antibody-drug conjugate. The conjugationmixture was gel filtered and loaded and eluted through a HiTrap S columnto remove excess drug-linker intermediate and other impurities.

FIG. 12 shows the general process to prepare a cysteine engineeredantibody expressed from cell culture for conjugation. When the cellculture media contains cysteine, disulfide adducts can form between thenewly introduced cysteine amino acid and cysteine from media. Thesecysteine adducts, depicted as a circle in the exemplary ThioMab (left)in FIG. 12, must be reduced to generate cysteine engineered antibodiesreactive for conjugation. Cysteine adducts, presumably along withvarious interchain disulfide bonds, are reductively cleaved to give areduced form of the antibody with reducing agents such as TCEP. Theinterchain disulfide bonds between paired cysteine residues are reformedunder partial oxidation conditions with copper sulfate, DHAA, orexposure to ambient oxygen. The newly introduced, engineered, andunpaired cysteine residues remain available for reaction with linkerreagents or drug-linker intermediates to form the antibody conjugates ofthe invention. The ThioMabs expressed in mammalian cell lines result inexternally conjugated Cys adduct to an engineered Cys through —S—S— bondformation. Hence the purified ThioMabs are treated with the reductionand reoxidation procedures as described in Example x to produce reactiveThioMabs. These ThioMabs are used to conjugate with maleimide containingcytotoxic drugs, fluorophores, and other labels.

Methods of Screening

Yet another embodiment of the present invention is directed to a methodof determining the presence of a CD22 polypeptide in a sample suspectedof containing the CD22 polypeptide, wherein the method comprisesexposing the sample to a cysteine engineered anti-CD22 antibody, orantibody drug conjugate thereof, that binds to the CD22 polypeptide anddetermining binding of the cysteine engineered anti-CD22 antibody, orantibody drug conjugate thereof, to the CD22 polypeptide in the sample,wherein the presence of such binding is indicative of the presence ofthe CD22 polypeptide in the sample. Optionally, the sample may containcells (which may be cancer cells) suspected of expressing the CD22polypeptide. The cysteine engineered anti-CD22 antibody, or antibodydrug conjugate thereof, employed in the method may optionally bedetectably labeled, attached to a solid support, or the like.

Another embodiment of the present invention is directed to a method ofdiagnosing the presence of a tumor in a mammal, wherein the methodcomprises (a) contacting a test sample comprising tissue cells obtainedfrom the mammal with a cysteine engineered anti-CD22 antibody, orantibody drug conjugate thereof, that binds to a CD22 polypeptide and(b) detecting the formation of a complex between the cysteine engineeredanti-CD22 antibody, or antibody drug conjugate thereof, and the CD22polypeptide in the test sample, wherein the formation of a complex isindicative of the presence of a tumor in the mammal. Optionally, thecysteine engineered anti-CD22 antibody, or antibody drug conjugatethereof, is detectably labeled, attached to a solid support, or thelike, and/or the test sample of tissue cells is obtained from anindividual suspected of having a cancerous tumor.

Metabolites of the Antibody-Drug Conjugates

Also falling within the scope of this invention are the in vivometabolic products of the ADC compounds described herein, to the extentsuch products are novel and unobvious over the prior art. Such productsmay result for example from the oxidation, reduction, hydrolysis,amidation, esterification, enzymatic cleavage, and the like, of theadministered compound. Accordingly, the invention includes novel andunobvious compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g. ¹⁴C or ³H) ADC, administering it parenterally in adetectable dose (e.g. greater than about 0.5 mg/kg) to an animal such asrat, mouse, guinea pig, monkey, or to man, allowing sufficient time formetabolism to occur (typically about 30 seconds to 30 hours) andisolating its conversion products from the urine, blood or otherbiological samples. These products are easily isolated since they arelabeled (others are isolated by the use of antibodies capable of bindingepitopes surviving in the metabolite). The metabolite structures aredetermined in conventional fashion, e.g. by MS, LC/MS or NMR analysis.In general, analysis of metabolites is done in the same way asconventional drug metabolism studies well-known to those skilled in theart. The conversion products, so long as they are not otherwise found invivo, are useful in diagnostic assays for therapeutic dosing of the ADCcompounds of the invention.

Pharmaceutical Formulations

Administration of Antibody-Drug Conjugates, Including Thio-Antibody DrugConjugates

The antibody-drug conjugates (ADC), including thio-antibody drugconjugates (TDC), of the invention may be administered by any routeappropriate to the condition to be treated. The ADC will typically beadministered parenterally, i.e. infusion, subcutaneous, intramuscular,intravenous, intradermal, intrathecal and epidural.

For treating these cancers, in one embodiment, the antibody-drugconjugate is administered via intravenous infusion. The dosageadministered via infusion is in the range of about 1 μg/m² to about10,000 μg/m² per dose, generally one dose per week for a total of one,two, three or four doses. Alternatively, the dosage range is of about 1μg/m² to about 1000 μg/m², about 1 μg/m² to about 800 μg/m², about 1μg/m² to about 600 μg/m², about 1 μg/m² to about 400 μg/m², about 10μg/m² to about 500 μg/m², about 10 μg/m² to about 300 μg/m², about 10μg/m² to about 200 μg/m², and about 1 μg/m² to about 200 μg/m². The dosemay be administered once per day, once per week, multiple times perweek, but less than once per day, multiple times per month but less thanonce per day, multiple times per month but less than once per week, onceper month or intermittently to relieve or alleviate symptoms of thedisease. Administration may continue at any of the disclosed intervalsuntil remission of the tumor or symptoms of the lymphoma, leukemia beingtreated. Administration may continue after remission or relief ofsymptoms is achieved where such remission or relief is prolonged by suchcontinued administration.

The invention also provides a method of alleviating an autoimmunedisease, comprising administering to a patient suffering from theautoimmune disease, a therapeutically effective amount of a humanized10F4 antibody-drug conjugate of any one of the preceding embodiments. Inpreferred embodiments the antibody is administered intravenously orsubcutaneously. The antibody-drug conjugate is administeredintravenously at a dosage in the range of about 1 μg/m² to about 100mg/m² per dose and in a specific embodiment, the dosage is 1 μg/m² toabout 500 μg/m². The dose may be administered once per day, once perweek, multiple times per week, but less than once per day, multipletimes per month but less than once per day, multiple times per month butless than once per week, once per month or intermittently to relieve oralleviate symptoms of the disease. Administration may continue at any ofthe disclosed intervals until relief from or alleviation of symptoms ofthe autoimmune disease being treated. Administration may continue afterrelief from or alleviation of symptoms is achieved where suchalleviation or relief is prolong by such continued administration.

The invention also provides a method of treating a B cell disordercomprising administering to a patient suffering from a B cell disorder,such as a B cell proliferative disorder (including without limitationlymphoma and leukemia) or an autoimmune disease, a therapeuticallyeffective amount of a humanized 10F4 antibody of any one of thepreceding embodiments, which antibody is not conjugated to a cytotoxicmolecule or a detectable molecule. The antibody will typically beadministered in a dosage range of about 1 μg/m² to about 1000 mg/m².

In one aspect, the invention further provides pharmaceuticalformulations comprising at least one anti-CD22 antibody of the inventionand/or at least one immunoconjugate thereof and/or at least oneanti-CD22 antibody-drug conjugate of the invention. In some embodiments,a pharmaceutical formulation comprises 1) an anti-CD22 antibody and/oran anti-CD22 antibody-drug conjugate and/or an immunoconjugate thereof,and 2) a pharmaceutically acceptable carrier. In some embodiments, apharmaceutical formulation comprises 1) an anti-CD22 antibody and/or animmunoconjugate thereof, and optionally, 2) at least one additionaltherapeutic agent.

Pharmaceutical formulations comprising an antibody or immunoconjugate ofthe invention or the antibody-drug conjugate of the invention areprepared for storage by mixing the antibody or antibody-drug conjugatehaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the formof aqueous solutions or lyophilized or other dried formulations.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, histidine and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride); phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Pharmaceutical formulations to be used for in vivo administration aregenerally sterile. This is readily accomplished by filtration throughsterile filtration membranes.

Active ingredients may also be entrapped in microcapsule prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody or immunoconjugate of theinvention, which matrices are in the form of shaped articles, e.g.,films, or microcapsule. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies or immunoconjugates remain in thebody for a long time, they may denature or aggregate as a result ofexposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Antibody-Drug Conjugate Treatments

It is contemplated that the antibody-drug conjugates (ADC) of thepresent invention may be used to treat various diseases or disorders,e.g. characterized by the overexpression of a tumor antigen. Exemplaryconditions or hyperproliferative disorders include benign or malignanttumors; leukemia and lymphoid malignancies. Others include neuronal,glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial,stromal, blastocoelic, inflammatory, angiogenic and immunologic,including autoimmune, disorders.

The ADC compounds which are identified in the animal models andcell-based assays can be further tested in tumor-bearing higher primatesand human clinical trials. Human clinical trials can be designed to testthe efficacy of the anti-CD22 monoclonal antibody or immunoconjugate ofthe invention in patients experiencing a B cell proliferative disorderincluding without limitation lymphoma, non-Hodgkins lymphoma (NHL),aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL),acute lymphocytic leukemia (ALL), and mantle cell lymphoma. The clinicaltrial may be designed to evaluate the efficacy of an ADC in combinationswith known therapeutic regimens, such as radiation and/or chemotherapyinvolving known chemotherapeutic and/or cytotoxic agents.

Generally, the disease or disorder to be treated is a hyperproliferativedisease such as a B cell proliferative disorder and/or a B cell cancer.Examples of cancer to be treated herein include, but are not limited to,B cell proliferative disorder is selected from lymphoma, non-Hodgkinslymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsedindolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.

The cancer may comprise CD22-expressing cells, such that the ADC of thepresent invention are able to bind to the cancer cells. To determineCD22 expression in the cancer, various diagnostic/prognostic assays areavailable. In one embodiment, CD22 overexpression may be analyzed byIHC. Parrafin-embedded tissue sections from a tumor biopsy may besubjected to the IHC assay and accorded a CD22 protein stainingintensity criteria with respect to the degree of staining and in whatproportion of tumor cells examined.

For the prevention or treatment of disease, the appropriate dosage of anADC will depend on the type of disease to be treated, as defined above,the severity and course of the disease, whether the molecule isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 ng/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 ng/kg to 100 mg/kg or more, depending onthe factors mentioned above. An exemplary dosage of ADC to beadministered to a patient is in the range of about 0.1 to about 10 mg/kgof patient weight.

For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of an anti-ErbB2 antibody.Other dosage regimens may be useful. The progress of this therapy iseasily monitored by conventional techniques and assays.

Combination Therapy

An antibody-drug conjugate (ADC) of the invention may be combined in apharmaceutical combination formulation, or dosing regimen as combinationtherapy, with a second compound having anti-cancer properties. Thesecond compound of the pharmaceutical combination formulation or dosingregimen preferably has complementary activities to the ADC of thecombination such that they do not adversely affect each other.

The second compound may be a chemotherapeutic agent, cytotoxic agent,cytokine, growth inhibitory agent, anti-hormonal agent, and/orcardioprotectant. Such molecules are suitably present in combination inamounts that are effective for the purpose intended. A pharmaceuticalcomposition containing an ADC of the invention may also have atherapeutically effective amount of a chemotherapeutic agent such as atubulin-forming inhibitor, a topoisomerase inhibitor, or a DNA binder.

In one aspect, the first compound is an anti-CD22 ADC of the inventionand the second compound is an anti-CD20 antibody (either a nakedantibody or an ADC). In one embodiment the second compound is ananti-CD20 antibody rituximab (Rituxan®) or 2H7 (Genentech, Inc., SouthSan Francisco, Calif.). Another antibodies useful for combinedimmunotherapy with anti-CD22 ADCs of the invention includes withoutlimitation, anti-VEGF (e.g, Avastin®).

Other therapeutic regimens may be combined with the administration of ananticancer agent identified in accordance with this invention, includingwithout limitation radiation therapy and/or bone marrow and peripheralblood transplants, and/or a cytotoxic agent, a chemotherapeutic agent,or a growth inhibitory agent. In one of such embodiments, achemotherapeutic agent is an agent or a combination of agents such as,for example, cyclophosphamide, hydroxydaunorubicin, adriamycin,doxorubincin, vincristine (Oncovin™), prednisolone, CHOP, CVP, or COP,or immunotherapeutics such as anti-CD20 (e.g., Rituxan®) or anti-VEGF(e.g., Avastin®). The combination therapy may be administered as asimultaneous or sequential regimen. When administered sequentially, thecombination may be administered in two or more administrations. Thecombined administration includes coadministration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities.

In one embodiment, treatment with an ADC involves the combinedadministration of an anticancer agent identified herein, and one or morechemotherapeutic agents or growth inhibitory agents, includingcoadministration of cocktails of different chemotherapeutic agents.Chemotherapeutic agents include taxanes (such as paclitaxel anddocetaxel) and/or anthracycline antibiotics. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturer's instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in “Chemotherapy Service”, (1992) Ed., M. C. Perry,Williams & Wilkins, Baltimore, Md.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other chemotherapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

Metabolites of the Antibody-Drug Conjugates

Also falling within the scope of this invention are the in vivometabolic products of the ADC compounds described herein, to the extentsuch products are novel and unobvious over the prior art. Such productsmay result for example from the oxidation, reduction, hydrolysis,amidation, esterification, enzymatic cleavage, and the like, of theadministered compound. Accordingly, the invention includes novel andunobvious compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g. 14C or 3H) ADC, administering it parenterally in adetectable dose (e.g. greater than about 0.5 mg/kg) to an animal such asrat, mouse, guinea pig, monkey, or to man, allowing sufficient time formetabolism to occur (typically about 30 seconds to 30 hours) andisolating its conversion products from the urine, blood or otherbiological samples. These products are easily isolated since they arelabeled (others are isolated by the use of antibodies capable of bindingepitopes surviving in the metabolite). The metabolite structures aredetermined in conventional fashion, e.g. by MS, LC/MS or NMR analysis.In general, analysis of metabolites is done in the same way asconventional drug metabolism studies well-known to those skilled in theart. The conversion products, so long as they are not otherwise found invivo, are useful in diagnostic assays for therapeutic dosing of the ADCcompounds of the invention.

Further Methods of Using Anti-CD22 Antibodies and Immunoconjugates

Diagnostic Methods and Methods of Detection

In one aspect, anti-CD22 antibodies and immunoconjugates of theinvention are useful for detecting the presence of CD22 in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue. In certain embodiments, such tissues includenormal and/or cancerous tissues that express CD22 at higher levelsrelative to other tissues, for example, B cells and/or B cell associatedtissues.

In one aspect, the invention provides a method of detecting the presenceof CD22 in a biological sample. In certain embodiments, the methodcomprises contacting the biological sample with an anti-CD22 antibodyunder conditions permissive for binding of the anti-CD22 antibody toCD22, and detecting whether a complex is formed between the anti-CD22antibody and CD22.

In one aspect, the invention provides a method of diagnosing a disorderassociated with increased expression of CD22. In certain embodiments,the method comprises contacting a test cell with an anti-CD22 antibody;determining the level of expression (either quantitatively orqualitatively) of CD22 by the test cell by detecting binding of theanti-CD22 antibody to CD22; and comparing the level of expression ofCD22 by the test cell with the level of expression of CD22 by a controlcell (e.g., a normal cell of the same tissue origin as the test cell ora cell that expresses CD22 at levels comparable to such a normal cell),wherein a higher level of expression of CD22 by the test cell ascompared to the control cell indicates the presence of a disorderassociated with increased expression of CD22. In certain embodiments,the test cell is obtained from an individual suspected of having adisorder associated with increased expression of CD22. In certainembodiments, the disorder is a cell proliferative disorder, such as acancer or a tumor.

Exemplary cell proliferative disorders that may be diagnosed using anantibody of the invention include a B cell disorder and/or a B cellproliferative disorder including, but not limited to, lymphoma,non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL,relapsed indolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.

In certain embodiments, a method of diagnosis or detection, such asthose described above, comprises detecting binding of an anti-CD22antibody to CD22 expressed on the surface of a cell or in a membranepreparation obtained from a cell expressing CD22 on its surface. Incertain embodiments, the method comprises contacting a cell with ananti-CD22 antibody under conditions permissive for binding of theanti-CD22 antibody to CD22, and detecting whether a complex is formedbetween the anti-CD22 antibody and CD22 on the cell surface. Anexemplary assay for detecting binding of an anti-CD22 antibody to CD22expressed CD22 on the surface of a cell is a “FACS” assay.

Certain other methods can be used to detect binding of anti-CD22antibodies to CD22. Such methods include, but are not limited to,antigen-binding assays that are well known in the art, such as westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, and immunohistochemistry (1HC).

In certain embodiments, anti-CD22 antibodies are labeled. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

In certain embodiments, anti-CD22 antibodies are immobilized on aninsoluble matrix. Immobilization entails separating the anti-CD22antibody from any CD22 that remains free in solution. Thisconventionally is accomplished by either insolubilizing the anti-CD22antibody before the assay procedure, as by adsorption to awater-insoluble matrix or surface (Bennich et al., U.S. Pat. No.3,720,760), or by covalent coupling (for example, using glutaraldehydecross-linking), or by insolubilizing the anti-CD22 antibody afterformation of a complex between the anti-CD22 antibody and CD22, e.g., byimmunoprecipitation.

Any of the above embodiments of diagnosis or detection may be carriedout using an immunoconjugate of the invention in place of or in additionto an anti-CD22 antibody.

Therapeutic Methods

An antibody or immunoconjugate of the invention may be used in, forexample, in vitro, ex vivo, and in vivo therapeutic methods. In oneaspect, the invention provides methods for inhibiting cell growth orproliferation, either in vivo or in vitro, the method comprisingexposing a cell to an anti-CD22 antibody or immunoconjugate thereofunder conditions permissive for binding of the immunoconjugate to CD22.“Inhibiting cell growth or proliferation” means decreasing a cell'sgrowth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 100%, and includes inducing cell death. In certainembodiments, the cell is a tumor cell. In certain embodiments, the cellis a B cell. In certain embodiments, the cell is a xenograft, e.g., asexemplified herein.

In one aspect, an antibody or immunoconjugate of the invention is usedto treat or prevent a B cell proliferative disorder. In certainembodiments, the cell proliferative disorder is associated withincreased expression and/or activity of CD22. For example, in certainembodiments, the B cell proliferative disorder is associated withincreased expression of CD22 on the surface of a B cell. In certainembodiments, the B cell proliferative disorder is a tumor or a cancer.Examples of B cell proliferative disorders to be treated by theantibodies or immunoconjugates of the invention include, but are notlimited to, lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL,relapsed aggressive NHL, relapsed indolent NHL, refractory NHL,refractory indolent NHL, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acutelymphocytic leukemia (ALL), and mantle cell lymphoma.

In one aspect, the invention provides methods for treating a B cellproliferative disorder comprising administering to an individual aneffective amount of an anti-CD22 antibody or immunoconjugate thereof. Incertain embodiments, a method for treating a B cell proliferativedisorder comprises administering to an individual an effective amount ofa pharmaceutical formulation comprising an anti-CD22 antibody oranti-CD22 immunoconjugate and, optionally, at least one additionaltherapeutic agent, such as those provided below. In certain embodiments,a method for treating a cell proliferative disorder comprisesadministering to an individual an effective amount of a pharmaceuticalformulation comprising 1) an immunoconjugate comprising an anti-CD22antibody and a cytotoxic agent; and optionally, 2) at least oneadditional therapeutic agent, such as those provided below.

In one aspect, at least some of the antibodies or immunoconjugates ofthe invention can bind CD22 from species other than human. Accordingly,antibodies or immunoconjugates of the invention can be used to bindCD22, e.g., in a cell culture containing CD22, in humans, or in othermammals having a CD22 with which an antibody or immunoconjugate of theinvention cross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgusand rhesus monkeys, pig or mouse). In one embodiment, an anti-CD22antibody or immunoconjugate can be used for targeting CD22 on B cells bycontacting the antibody or immunoconjugate with CD22 to form an antibodyor immunoconjugate-antigen complex such that a conjugated cytotoxin ofthe immunoconjugate accesses the interior of the cell. In oneembodiment, the CD22 is human CD22.

In one embodiment, an anti-CD22 antibody or immunoconjugate can be usedin a method for binding CD22 in an individual suffering from a disorderassociated with increased CD22 expression and/or activity, the methodcomprising administering to the individual the antibody orimmunoconjugate such that CD22 in the individual is bound. In oneembodiment, the bound antibody or immunoconjugate is internalized intothe B cell expressing CD22. In one embodiment, the CD22 is human CD22,and the individual is a human individual. Alternatively, the individualcan be a mammal expressing CD22 to which an anti-CD22 antibody binds.Still further the individual can be a mammal into which CD22 has beenintroduced (e.g., by administration of CD22 or by expression of atransgene encoding CD22).

An anti-CD22 antibody or immunoconjugate can be administered to a humanfor therapeutic purposes. Moreover, an anti-CD22 antibody orimmunoconjugate can be administered to a non-human mammal expressingCD22 with which the antibody cross-reacts (e.g., a primate, pig, rat, ormouse) for veterinary purposes or as an animal model of human disease.Regarding the latter, such animal models may be useful for evaluatingthe therapeutic efficacy of antibodies or immunoconjugates of theinvention (e.g., testing of dosages and time courses of administration).

Antibodies or immunoconjugates of the invention can be used either aloneor in combination with other compositions in a therapy. For instance, anantibody or immunoconjugate of the invention may be co-administered withat least one additional therapeutic agent and/or adjuvant. In certainembodiments, an additional therapeutic agent is a cytotoxic agent, achemotherapeutic agent, or a growth inhibitory agent. In one of suchembodiments, a chemotherapeutic agent is an agent or a combination ofagents such as, for example, cyclophosphamide, hydroxydaunorubicin,adriamycin, doxorubincin, vincristine (Oncovin™), prednisolone, CHOP,CVP, or COP, or immunotherapeutics such as anti-CD20 (e.g., Rituxan®) oranti-VEGF (e.g., Avastin®), wherein the combination therapy is useful inthe treatment of cancers and/or B cell disorders such as B cellproliferative disorders including lymphoma, non-Hodgkins lymphoma (NHL),aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL),acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody or immunoconjugate of the invention canoccur prior to, simultaneously, and/or following, administration of theadditional therapeutic agent and/or adjuvant. Antibodies orimmunoconjugates of the invention can also be used in combination withradiation therapy.

An antibody or immunoconjugate of the invention (and any additionaltherapeutic agent or adjuvant) can be administered by any suitablemeans, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody orimmunoconjugate is suitably administered by pulse infusion, particularlywith declining doses of the antibody or immunoconjugate. Dosing can beby any suitable route, e.g. by injections, such as intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic.

Antibodies or immunoconjugates of the invention would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody or immunoconjugate need notbe, but is optionally formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of antibody or immunoconjugatepresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody or immunoconjugate of the invention (when used alone or incombination with one or more other additional therapeutic agents, suchas chemotherapeutic agents) will depend on the type of disease to betreated, the type of antibody or immunoconjugate, the severity andcourse of the disease, whether the antibody or immunoconjugate isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody orimmunoconjugate, and the discretion of the attending physician. Theantibody or immunoconjugate is suitably administered to the patient atone time or over a series of treatments. Depending on the type andseverity of the disease, about 1 μg/kg to 100 mg/kg (e.g. 0.1 mg/kg-20mg/kg) of antibody or immunoconjugate can be an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the antibody or immunoconjugate would bein the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or moredoses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or anycombination thereof) of antibody or immunoconjugate may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of the antibodyor immunoconjugate). An initial higher loading dose, followed by one ormore lower doses may be administered. An exemplary dosing regimencomprises administering an initial loading dose of about 4 mg/kg,followed by a weekly maintenance dose of about 2 mg/kg of the antibody.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

Assays

Anti-CD22 antibodies and immunoconjugates of the invention may becharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

Activity Assays

In one aspect, assays are provided for identifying anti-CD22 antibodiesor immunoconjugates thereof having biological activity. Biologicalactivity may include, e.g., the ability to inhibit cell growth orproliferation (e.g., “cell killing” activity), or the ability to inducecell death, including programmed cell death (apoptosis). Antibodies orimmunoconjugates having such biological activity in vivo and/or in vitroare also provided.

In certain embodiments, an anti-CD22 antibody or immunoconjugate thereofis tested for its ability to inhibit cell growth or proliferation invitro. Assays for inhibition of cell growth or proliferation are wellknown in the art. Certain assays for cell proliferation, exemplified bythe “cell killing” assays described herein, measure cell viability. Onesuch assay is the CellTiter-Glo™ Luminescent Cell Viability Assay, whichis commercially available from Promega (Madison, Wis.). That assaydetermines the number of viable cells in culture based on quantitationof ATP present, which is an indication of metabolically active cells.See Crouch et al (1993) J. Immunol. Meth. 160:81-88, U.S. Pat. No.6,602,677. The assay may be conducted in 96- or 384-well format, makingit amenable to automated high-throughput screening (HTS). See Cree et al(1995) AntiCancer Drugs 6:398-404. The assay procedure involves adding asingle reagent (CellTiter-Glo® Reagent) directly to cultured cells. Thisresults in cell lysis and generation of a luminescent signal produced bya luciferase reaction. The luminescent signal is proportional to theamount of ATP present, which is directly proportional to the number ofviable cells present in culture. Data can be recorded by luminometer orCCD camera imaging device. The luminescence output is expressed asrelative light units (RLU).

Another assay for cell proliferation is the “MTT” assay, a colorimetricassay that measures the oxidation of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazanby mitochondrial reductase. Like the CellTiter-Glo™ assay, this assayindicates the number of metabolically active cells present in a cellculture. See, e.g., Mosmann (1983) J. Immunol. Meth. 65:55-63, and Zhanget al. (2005) Cancer Res. 65:3877-3882.

In one aspect, an anti-CD22 antibody is tested for its ability to inducecell death in vitro. Assays for induction of cell death are well knownin the art. In some embodiments, such assays measure, e.g., loss ofmembrane integrity as indicated by uptake of propidium iodide (PI),trypan blue (see Moore et al. (1995) Cytotechnology, 17:1-11), or 7AAD.In an exemplary PI uptake assay, cells are cultured in Dulbecco'sModified Eagle Medium (D-MEM):Ham's F-12 (50:50) supplemented with 10%heat-inactivated FBS (Hyclone) and 2 mM L-glutamine. Thus, the assay isperformed in the absence of complement and immune effector cells. Cellsare seeded at a density of 3×10⁶ per dish in 100×20 mm dishes andallowed to attach overnight. The medium is removed and replaced withfresh medium alone or medium containing various concentrations of theantibody or immunoconjugate. The cells are incubated for a 3-day timeperiod. Following treatment, monolayers are washed with PBS and detachedby trypsinization. Cells are then centrifuged at 1200 rpm for 5 minutesat 4° C., the pellet resuspended in 3 ml cold Ca²⁺ binding buffer (10 mMHepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mmstrainer-capped 12×75 mm tubes (1 ml per tube, 3 tubes per treatmentgroup) for removal of cell clumps. Tubes then receive PI (10 μg/ml).Samples are analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™CellQuest software (Becton Dickinson). Antibodies or immunoconjugateswhich induce statistically significant levels of cell death asdetermined by PI uptake are thus identified.

In one aspect, an anti-CD22 antibody or immunoconjugate is tested forits ability to induce apoptosis (programmed cell death) in vitro. Anexemplary assay for antibodies or immunconjugates that induce apoptosisis an annexin binding assay. In an exemplary annexin binding assay,cells are cultured and seeded in dishes as discussed in the precedingparagraph. The medium is removed and replaced with fresh medium alone ormedium containing 0.001 to 10 μg/ml of the antibody or immunoconjugate.Following a three-day incubation period, monolayers are washed with PBSand detached by trypsinization. Cells are then centrifuged, resuspendedin Ca²⁺ binding buffer, and aliquoted into tubes as discussed in thepreceding paragraph. Tubes then receive labeled annexin (e.g. annexinV-FITC) (1 μg/ml). Samples are analyzed using a FACSCAN™ flow cytometerand FACSCONVERT™ CellQuest software (BD Biosciences). Antibodies orimmunoconjugates that induce statistically significant levels of annexinbinding relative to control are thus identified. Another exemplary assayfor antibodies or immunconjugates that induce apoptosis is a histone DNAELISA colorimetric assay for detecting internucleosomal degradation ofgenomic DNA. Such an assay can be performed using, e.g., the Cell DeathDetection ELISA kit (Roche, Palo Alto, Calif.).

Cells for use in any of the above in vitro assays include cells or celllines that naturally express CD22 or that have been engineered toexpress CD22. Such cells include tumor cells that overexpress CD22relative to normal cells of the same tissue origin. Such cells alsoinclude cell lines (including tumor cell lines) that express CD22 andcell lines that do not normally express CD22 but have been transfectedwith nucleic acid encoding CD22.

In one aspect, an anti-CD22 antibody or immunoconjugate thereof istested for its ability to inhibit cell growth or proliferation in vivo.In certain embodiments, an anti-CD22 antibody or immunoconjugate thereofis tested for its ability to inhibit tumor growth in vivo. In vivo modelsystems, such as xenograft models, can be used for such testing. In anexemplary xenograft system, human tumor cells are introduced into asuitably immunocompromised non-human animal, e.g., a SCID mouse. Anantibody or immunoconjugate of the invention is administered to theanimal. The ability of the antibody or immunoconjugate to inhibit ordecrease tumor growth is measured. In certain embodiments of the abovexenograft system, the human tumor cells are tumor cells from a humanpatient. Such cells useful for preparing xenograft models include humanleukemia and lymphoma cell lines, which include without limitation theBJAB-luc cells (an EBV-negative Burkitt's lymphoma cell line transfectedwith the luciferase reporter gene), Ramos cells (ATCC, Manassas, Va.,CRL-1923), Raji cells (ATCC, Manassas, Va., CCL-86), SuDHL-4 cells(DSMZ, Braunschweig, Germany, AAC 495), DoHH2 cells (seeKluin-Neilemans, H. C. et al., Leukemia 5:221-224 (1991), andKluin-Neilemans, H. C. et al., Leukemia 8:1385-1391 (1994)), Granta-519cells (see Jadayel, D. M. et al, Leukemia 11(1):64-72 (1997)). Incertain embodiments, the human tumor cells are introduced into asuitably immunocompromised non-human animal by subcutaneous injection orby transplantation into a suitable site, such as a mammary fat pad.

Binding Assays and Other Assays

In one aspect, an anti-CD22 antibody is tested for its antigen bindingactivity. For example, in certain embodiments, an anti-CD22 antibody istested for its ability to bind to CD22 expressed on the surface of acell. A FACS assay may be used for such testing.

In one aspect, competition assays may be used to identify a monoclonalantibody that competes with murine 10F4.4.1 antibody, humanized 10F4v1antibody, humanized 10F4v3 antibody and/or murine 5E8.1.8 antibody forbinding to CD22. In certain embodiments, such a competing antibody bindsto the same epitope (e.g., a linear or a conformational epitope) that isbound by murine 10F4.4.1 antibody, humanized 10F4v1 antibody, humanized10F4v3 antibody and/or murine 5E8.1.8 antibody. Exemplary competitionassays include, but are not limited to, routine assays such as thoseprovided in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Detailedexemplary methods for mapping an epitope to which an antibody binds areprovided in Morris (1996) “Epitope Mapping Protocols,” in Methods inMolecular Biology vol. 66 (Humana Press, Totowa, N.J.). Two antibodiesare said to bind to the same epitope if each blocks binding of the otherby 50% or more.

In an exemplary competition assay, immobilized CD22 is incubated in asolution comprising a first labeled antibody that binds to CD22 (e.g.,murine 10F4.4.1 antibody, humanized 10F4v1 antibody, humanized 10F4v3antibody and/or murine 5E8.1.8 antibody) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to CD22. The second antibody may be present in a hybridomasupernatant. As a control, immobilized CD22 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to CD22, excess unbound antibody is removed, and theamount of label associated with immobilized CD22 is measured. If theamount of label associated with immobilized CD22 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to CD22. In certain embodiments, immobilized CD22 is presenton the surface of a cell or in a membrane preparation obtained from acell expressing CD22 on its surface.

In one aspect, purified anti-CD22 antibodies can be furthercharacterized by a series of assays including, but not limited to,N-terminal sequencing, amino acid analysis, non-denaturing sizeexclusion high pressure liquid chromatography (HPLC), mass spectrometry,ion exchange chromatography and papain digestion.

In one embodiment, the invention contemplates an altered antibody thatpossesses some but not all effector functions, which make it a desirablecandidate for many applications in which the half life of the antibodyin vivo is important yet certain effector functions (such as complementand ADCC) are unnecessary or deleterious. In certain embodiments, the Fcactivities of the antibody are measured to ensure that only the desiredproperties are maintained. In vitro and/or in vivo cytotoxicity assayscan be conducted to confirm the reduction/depletion of CDC and/or ADCCactivities. For example, Fc receptor (FcR) binding assays can beconducted to ensure that the antibody lacks FcγR binding (hence likelylacking ADCC activity), but retains FcRn binding ability. The primarycells for mediating ADCC, NK cells, express FcγRIII only, whereasmonocytes express FcγRI, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-92 (1991). An example of an in vitroassay to assess ADCC activity of a molecule of interest is described inU.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for suchassays include peripheral blood mononuclear cells (PBMC) and NaturalKiller (NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).C1q binding assays may also be carried out to confirm that the antibodyis unable to bind C1q and hence lacks CDC activity. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed. FcRn binding and invivo clearance/half life determinations can also be performed usingmethods known in the art.

Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1 Preparation of Murine Anti-Human CD22 Monoclonal Antibody

Murine monoclonal antibodies capable of specifically binding human CD22was prepared. BALB/c female mice, age six weeks, were immunized in theirfoot pads with purified human CD22 his-8 tagged extracellular domainlacking domains 3 and 4 (SEQ ID NO:30 (ECD) plus the sequenceGRAHHHHHHHH at the C-terminus) or CD22 his-8 tagged extracellular domaincomprising domains 1-7 (SEQ ID NO:28 (ECD) plus the above His sequencetag) in Ribi's adjuvant. Subsequent injections were performed in thesame manner at one and three weeks after the initial immunizations.Three days after the final injection, the inguinal and popliteal lymphnodes were removed and pooled, and a single cell suspension was made bypassing the tissue through steel gauze. The cells were fused at a 4:1ratio with mouse myeloma such as P3X63-Ag8.653 (ATCC CRL 1580) in highglucose (DMEM) containing 50% w/v polyethylene glycol 4000. The fusedcells were then plated at a density of 2×105 per well in 96 well tissueculture plates. After 24 hours HAT selective medium(hypoxanthine/aminopterin/thymidine, Sigma, #H0262) was added. Fifteendays after the fusion, supernatants of growing cells were tested for thepresence of antibodies specific for human CD22 using an enzyme-linkedimmunosorbent assay (ELISA).

The murine anti-human CD22 10F4.4.1 (mu 10F4) and 5E8.1.8 (mu 5E8)monoclonal antibodies were selected for further study based oncell-based assays and plate assays which showed the antibodies to bindspecifically to human CD22. The assays are described in the followingparagraphs.

ELISA-based assays: Anti-CD22 antibody screening by ELISA is performedas follows, with all incubations done at room temperature. Test plates(Nunc Immunoplate) were coated for 2 hours with purified CD22 in 50 mMsodium carbonate buffer, pH 9.6, then blocked with 0.5% bovine serumalbumin in phosphate buffered saline (PBS) for 30 minutes, then washedfour times with PBS containing 0.05% Tween 20 (PBST). Test antibodysupernatants are added and incubated two hours with shaking, then washedfour times with PBST. The plates are developed by adding 100 μl/well ofa solution containing 10 mg of o-phenylenediamine dihydrochloride(Sigma, #P8287) and 10 l of a 30% hydrogen peroxide solution in 25 mlphosphate citrate buffer, pH 5.0, and incubating for 15 minutes. Thereaction is stopped by adding 100 μl/well of 2.5 M sulfuric acid. Datais obtained by reading the plates in an automated ELISA plate reader atan absorbance of 490 nm.

Example 2

FACS Based Assays for Analysis of Ant-Human CD22 Monoclonal Antibodies(MAbs).

CHO cells expressing human CD22 on their surface were incubated withanti-CD22 hybridoma supernatant in 100 μl FACS buffer (0.1% BSA, 10 mMsodium azide in PBS, pH 7.4) for 30 minutes at 4° C. followed by onewash with FACS buffer. The amount of anti-CD22 binding was determined byincubating an aliquot of the antibody/cell mixture with a polyclonalFITC conjugated goat or rabbit anti-mouse IgG (Accurate Chem. Co.,Westbury, N.Y.) (for murine test antibodies) or goat or rabbitanti-human IgG (for humanized antibodies) for 30 minutes at 4° C.followed by three washes with FACS buffer.

Example 3 Preparation of Humanized Anti-CD22 Antibodies

Humanized 10F4 antibodies were generated wherein hypervariable region(HVR) amino acid residues (interchangeably referred to ascomplementarity determining regions or CDRs) were modified viasite-directed mutagenesis (Kunkel et al., Methods Enzymol. (1987),154:367-382) to arrive at two variants, humanized 10F4v1 and humanized10F4v2 (also referred to herein as “10F4v1,” “hu10F4v1,” “10F4v2,” or“hu10F4v2,” respectively). A third version, humanized 10F4v3 (“10F4v3”or “hu10Fv3”), used in some studies disclosed herein has the same lightand heavy chain amino acid sequences for the mature protein as hu10F4v2,but comprises a different signal sequence in the vector used for proteinexpression.

Humanization of the murine 10F4 antibody was preformed as disclosedherein. Briefly, the hypervariable regions of the light and heavy chainsof murine 10F4 were cloned into modified consensus framework sequencesto generate the light and heavy chain variable regions amino acidsequences shown in FIGS. 2A and 2B. Alternative light and heavy chainframework sequences that may be used as framework sequences ofantibodies of the invention are shown in FIGS. 3 and 4.

A monovalent Fab-g3 display vector (pV0350-2B) phagemid having two openreading frames under control of the phoA promoter, essentially asdescribed in Lee et al., J. Mol. Biol. 340:1073-93 (2004), was used inthe humanization of the 10F4 antibody. The first open reading framecomprised the E. coli heat stable STII signal sequence for proteinsecretion fused to the VL and CH1 domains of the acceptor light chainsequence. The second open reading frame comprised the STII signalsequence fused to the VH and CH1 domains of the acceptor heavy chainsequence followed by a truncated minor phage coat protein P3.

The VH and VL domains from murine 10F4 (SEQ ID NOs:89 and 90,respectively) were aligned with the human subgroup III consensus VH(huIII) domain (SEQ ID NO:24) and human consensus kappal (huK1) domain(SEQ ID NO:25), respectively. The amino acid sequences of thehypervariable regions (HVRs, interchangeably referred to herein ascomplimentary determining regions (CDRs)) of the murine anti-human CD22MAb 10F4 were inserted into consensus framework sequences as follows.The light chain HVRs (HVR-L1 (Kabat positions 24-34), HVR-L2 (Kabatpositions 50-56), and HVR-L3 (Kabat positions 89-97) of the mu 10F4antibody were engineered into a human kappa I (huKI) consensus sequenceantibody framework to produce the humanized 10F4v1 light chain (SEQ IDNO:17, FIG. 2B). The heavy chain HVRs (HVR-H1 (Kabat positions 26-35),HVR-H2 (Kabat positions 49-65), and HVR-H3 (Kabat positions 95-102) ofthe mu 10F4 antibody were engineered into a modified human subgroup III(humIII) consensus VH domain which differs from the humIII sequence atthree positions: R71A, N73T, and L78A were used (see Carter et all,Proc. Natl. Acad. Sci. USA 89:4285 (1992)) to produce the humanized10F4v1 heavy chain variable region (SEQ ID NO:16, FIG. 2A). Geneticengineering of HVRs into the acceptor frameworks was performed by Kunkelmutagenesis using a separate oligonucleotide for each hypervariableregion. The sequence of each clone was determined by standard DNAsequencing techniques. Hypervariable regions and framework regions shownin FIGS. 2A and 2B are numbered according to Kabat numbering (Kabat etal. (1991), supra). The light and heavy chains were sequenced and theamino acid sequences of the variable regions (including HVRs andframework regions (FRs)) of the huKI, the humIII, murine 10F4, humanized10F4v1 and humanized 10F4v2 are shown in FIGS. 2A and 2B. Humanized10F4v3 antibody has the identical amino acid sequence as 10F4v2.

Nucleic acid molecules encoding amino acid sequence variants of theantibody, antibody fragment, VL domain or VH domain are prepared by avariety of methods known in the art. These methods include, but are notlimited to, isolation from a natural source (in the case of naturallyoccurring amino acid sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the antibody, antibody fragment, VL domain orVH domain. For example, libraries can be created by targeting VLaccessible amino acid positions in VH, and optionally in one or moreCDRs, for amino acid substitution with variant amino acids using theKunkel method. See, for e.g., Kunkel et al., Methods Enzymol. (1987),154:367-382 and the examples herein. Generation of randomized sequencesis also described below in the Examples.

The sequence of oligonucleotides includes one or more of the designedcodon sets for a particular position in a CDR(HVR) or FR region of apolypeptide of the invention. A codon set is a set of differentnucleotide triplet sequences used to encode desired variant amino acids.Codon sets can be represented using symbols to designate particularnucleotides or equimolar mixtures of nucleotides as shown in belowaccording to the IUB code.

IUB CODES G Guanine A Adenine T Thymine C Cytosine R (A or G) Y (C or T)M (A or C) K (G or T) S (C or G) W (A or T) H (A or C or T) B (C or G orT) V (A or C or G) D (A or G or T) N (A or C or G or T)

For example, in the codon set DVK, D can be nucleotides A or G or T; Vcan be A or G or C; and K can be G or T. This codon set can present 18different codons and can encode amino acids Ala, Trp, Tyr, Lys, Thr,Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

Oligonucleotide or primer sets can be synthesized using standardmethods. A set of oligonucleotides can be synthesized, for example, bysolid phase synthesis, containing sequences that represent all possiblecombinations of nucleotide triplets provided by the codon set and thatwill encode the desired group of amino acids. Synthesis ofoligonucleotides with selected nucleotide “degeneracy” at certainpositions is well known in that art. Such sets of nucleotides havingcertain codon sets can be synthesized using commercial nucleic acidsynthesizers (available from, for example, Applied Biosystems, FosterCity, Calif.), or can be obtained commercially (for example, from LifeTechnologies, Rockville, Md.). Therefore, a set of oligonucleotidessynthesized having a particular codon set will typically include aplurality of oligonucleotides with different sequences, the differencesestablished by the codon set within the overall sequence.Oligonucleotides, as used according to the invention, have sequencesthat allow for hybridization to a variable domain nucleic acid templateand also can include restriction enzyme sites for cloning purposes.

In one method, nucleic acid sequences encoding variant amino acids canbe created by oligonucleotide-mediated mutagenesis. This technique iswell known in the art as described by Zoller et al, 1987, Nucleic AcidsRes. 10:6487-6504. Briefly, nucleic acid sequences encoding variantamino acids are created by hybridizing an oligonucleotide set encodingthe desired codon sets to a DNA template, where the template is thesingle-stranded form of the plasmid containing a variable region nucleicacid template sequence. After hybridization, DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will contain thecodon sets as provided by the oligonucleotide set.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation(s). This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Nat'l. Acad. Sci. USA, 75:5765 (1978).

The DNA template is generated by those vectors that are either derivedfrom bacteriophage M13 vectors (the commercially available M13 mp18 andM13 mp19 vectors are suitable), or those vectors that contain asingle-stranded phage origin of replication as described by Viera etal., Meth. Enzymol., 153:3 (1987). Thus, the DNA that is to be mutatedcan be inserted into one of these vectors in order to generatesingle-stranded template. Production of the single-stranded template isdescribed in sections 4.21-4.41 of Sambrook et al., above.

To alter the native DNA sequence, the oligonucleotide is hybridized tothe single stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually T7 DNA polymerase or the Klenowfragment of DNA polymerase I, is then added to synthesize thecomplementary strand of the template using the oligonucleotide as aprimer for synthesis. A heteroduplex molecule is thus formed such thatone strand of DNA encodes the mutated form of gene 1, and the otherstrand (the original template) encodes the native, unaltered sequence ofgene 1. This heteroduplex molecule is then transformed into a suitablehost cell, usually a prokaryote such as E. coli JM101. After growing thecells, they are plated onto agarose plates and screened using theoligonucleotide primer radiolabelled with a 32-Phosphate to identify thebacterial colonies that contain the mutated DNA.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: The singlestranded oligonucleotide is annealed to the single-stranded template asdescribed above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTT), is combined with a modifiedthiodeoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham). This mixture is added to the template-oligonucleotidecomplex. Upon addition of DNA polymerase to this mixture, a strand ofDNA identical to the template except for the mutated bases is generated.In addition, this new strand of DNA will contain dCTP-(aS) instead ofdCTP, which serves to protect it from restriction endonucleasedigestion. After the template strand of the double-stranded heteroduplexis nicked with an appropriate restriction enzyme, the template strandcan be digested with ExoIII nuclease or another appropriate nucleasepast the region that contains the site(s) to be mutagenized. Thereaction is then stopped to leave a molecule that is only partiallysingle-stranded. A complete double-stranded DNA homoduplex is thenformed using DNA polymerase in the presence of all fourdeoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplexmolecule can then be transformed into a suitable host cell.

As indicated previously the sequence of the oligonucleotide set is ofsufficient length to hybridize to the template nucleic acid and mayalso, but does not necessarily, contain restriction sites. The DNAtemplate can be generated by those vectors that are either derived frombacteriophage M13 vectors or vectors that contain a single-strandedphage origin of replication as described by Viera et al. ((1987) Meth.Enzymol., 153:3). Thus, the DNA that is to be mutated must be insertedinto one of these vectors in order to generate single-stranded template.Production of the single-stranded template is described in sections4.21-4.41 of Sambrook et al., supra.

According to another method, a library can be generated by providingupstream and downstream oligonucleotide sets, each set having aplurality of oligonucleotides with different sequences, the differentsequences established by the codon sets provided within the sequence ofthe oligonucleotides. The upstream and downstream oligonucleotide sets,along with a variable domain template nucleic acid sequence, can be usedin a polymerase chain reaction to generate a “library” of PCR products.The PCR products can be referred to as “nucleic acid cassettes”, as theycan be fused with other related or unrelated nucleic acid sequences, forexample, viral coat proteins and dimerization domains, using establishedmolecular biology techniques.

Oligonucleotide sets can be used in a polymerase chain reaction using avariable domain nucleic acid template sequence as the template to createnucleic acid cassettes. The variable domain nucleic acid templatesequence can be any portion of the heavy immunoglobulin chainscontaining the target nucleic acid sequences (ie., nucleic acidsequences encoding amino acids targeted for substitution). The variableregion nucleic acid template sequence is a portion of a double strandedDNA molecule having a first nucleic acid strand and complementary secondnucleic acid strand. The variable domain nucleic acid template sequencecontains at least a portion of a variable domain and has at least oneCDR. In some cases, the variable domain nucleic acid template sequencecontains more than one CDR. An upstream portion and a downstream portionof the variable domain nucleic acid template sequence can be targetedfor hybridization with members of an upstream oligonucleotide set and adownstream oligonucleotide set.

A first oligonucleotide of the upstream primer set can hybridize to thefirst nucleic acid strand and a second oligonucleotide of the downstreamprimer set can hybridize to the second nucleic acid strand. Theoligonucleotide primers can include one or more codon sets and bedesigned to hybridize to a portion of the variable region nucleic acidtemplate sequence. Use of these oligonucleotides can introduce two ormore codon sets into the PCR product (ie., the nucleic acid cassette)following PCR. The oligonucleotide primer that hybridizes to regions ofthe nucleic acid sequence encoding the antibody variable domain includesportions that encode CDR residues that are targeted for amino acidsubstitution.

The upstream and downstream oligonucleotide sets can also be synthesizedto include restriction sites within the oligonucleotide sequence. Theserestriction sites can facilitate the insertion of the nucleic acidcassettes (i.e., PCR reaction products) into an expression vector havingadditional antibody sequence. In one embodiment, the restriction sitesare designed to facilitate the cloning of the nucleic acid cassetteswithout introducing extraneous nucleic acid sequences or removingoriginal CDR or framework nucleic acid sequences.

Nucleic acid cassettes can be cloned into any suitable vector forexpression of a portion or the entire light or heavy chain sequencecontaining the targeted amino acid substitutions generated via the PCRreaction. According to methods detailed in the invention, the nucleicacid cassette is cloned into a vector allowing production of a portionor the entire light or heavy chain sequence fused to all or a portion ofa viral coat protein (i.e., creating a fusion protein) and displayed onthe surface of a particle or cell. While several types of vectors areavailable and may be used to practice this invention, phagemid vectorsare the preferred vectors for use herein, as they may be constructedwith relative ease, and can be readily amplified. Phagemid vectorsgenerally contain a variety of components including promoters, signalsequences, phenotypic selection genes, origin of replication sites, andother necessary components as are known to those of ordinary skill inthe art.

When a particular variant amino acid combination is to be expressed, thenucleic acid cassette contains a sequence that is able to encode all ora portion of the heavy or light chain variable domain, and is able toencode the variant amino acid combinations. For production of antibodiescontaining these variant amino acids or combinations of variant aminoacids, as in a library, the nucleic acid cassettes can be inserted intoan expression vector containing additional antibody sequence, forexample all or portions of the variable or constant domains of the lightand heavy chain variable regions. These additional antibody sequencescan also be fused to other nucleic acids sequences, such as sequencesthat encode viral coat proteins and therefore allow production of afusion protein.

Example 4 Variable Region Sequence Determination

The nucleic acid and amino acid sequences of the murine and humanized10F4 monoclonal antibodies were determined by standard procedures. TotalRNA was extracted from hybridoma cells producing the mouse anti-humanCD22 10F4.4.1 monoclonal antibodies using the RNeasy® Mini Kit (Qiagen,Germany). The variable light (VL) and variable heavy (VH) domains wereamplified using RT-PCR with degenerate primers. The forward primers werespecific for the N-terminal amino acid sequences of the VL and VHregions of the antibody. Respectively, the light chain and heavy chainreverse primers were designed to anneal to a region in the constantlight (CL) and constant heavy domain 1 (CH₁), which is highly conservedacross species. Amplified VH and VL were cloned into a pRK mammaliancell expression vector (Shields et al., J. Biol. Chem. 276:659-04(2000)). The polynucleotide sequence of the inserts was determined usingroutine sequencing methods. The amino acid sequences of the murinechimeric 10F4 and humanized 10F4v1 and humanized 10F4v2 light and heavychain variable regions are shown in FIGS. 2A and 2B.

Humanized 10F4v1 was further modified at HVR-L1 position 28 (N28) (SEQID NO:9) (see FIG. 2B. The asparagine residue at that position wasreplaced with a valine residue (N28V) to generate HVR-L1 (SEQ ID NO:10)for the hu10F4v2 and hu10F4v3 variants, which showed improved bindingaffinity. These variants comprise the same variable and constant domainsequences of the mature antibody and differ only in a signal sequencenot found in the mature antibody of the invention.

Additional amino acid sequence modifications were generated at one orboth of amino acids Asn28 (N28) and/or Asn30 (N30) of the HVR-L1hypervariable region (see FIG. 2B) of hu10F4v1. Because N28 and N30 arepossible sites for deamination, amino acid changes at these sites weretested. For example, Asparagine at position 28 (N28) was replacedalternatively with A, Q, S, D, V, or I, and Asparagine at position 30(N30) was replaced alternatively with A or Q. Amino acid sequencechanges in the HVR-L1 domain according to the invention are provided inTable 2 along with their binding affinities as tested by competitionanalysis in a phage ELISA assay (IC50) using standard procedures.

TABLE 2 Substitution Variants of hu 10F4v1 antibody Amino Acid Change InHVR-L1 HVR-L1 SEQ ID Binding Affinity FIG. 2B NO (nM) No change 9 8(N28, N30) N28A, N30 19 8 N28Q, N30 20 7.3 N28S, N30 21 12 N28D, N30 2212 N28V, N30 10 7.3 N28I, N30 23 9.8 N28, N30A 32 7.7 N28, N30Q 33 10

For generation of full-length human IgG1 versions of humanized 10F4antibody, the heavy and light chains are subcloned separately intopreviously described pRK plasmids (Gorman, C. M. et al. (1990), DNAProtein Eng. Tech. 2: 3). Appropriate heavy and light chain plasmids(depending upon the sequence change(s) desired) are cotransfected intoan adenovirus-transformed human embryonic kidney cell line, known as 293(Graham, F. L. et al. (1977), J. Gen. Virol. 36: 59), using a highefficiency procedure (Graham et al., supra & Gorman, C. M., Science 221:551). Media is changed to serum free and harvested daily for up to 5days. Antibodies are purified from the pooled supernatants using proteinA-Sepharose CL-4B (Pharmacia). The eluted antibody is buffer exchangedinto PBS by G25 gel filtration, concentrated by ultrafiltration using aCentriprep-30 or Centricon-100 (Millipore), and stored at 4° C. Theconcentration of antibody is determined using total IgG-binding ELISA.

Exemplary heavy chain IgG1 constant domains according to the inventionare depicted in FIG. 5A. An exemplary human light chain κ constantdomain comprises, for example,RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHDVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:37). Thefull length amino acid sequence of h10F4v2 is shown in FIG. 5B in whichthe constant regions of the light and heavy chains are indicated byunderlining. The h10F4v1, v2, and v3 antibodies are IgG1 isotype.

Characterization of Anti-CD22 Antibodies

Example 5 Epitope Mapping

The epitopes of CD22 to which 10F4.4.1 and 5E8.1.8 antibodies bound weredetermined according to the following procedures. CD22 sequences lackingvarious of the seven immunoglobulin-like domains of the major CD22isoform (CD22beta) were cloned and transformed into cells for stableexpression. For example, CD22 variants lacking domain 1 (Δ1), domain 2(Δ2), or domains 3 and 4 (Δ3,4) were cloned, transformed into CHO cells,and expressed on the cells. Control cells expressed CD22beta. Deletionswere performed using Stratagene QuikChange XL™ reagent kit. Deletion ofdomain 1 was performed by deletion of amino acids 22-138; deletion ofdomain 2 was performed by deletion of amino acids 139-242; and deleteddomains 3 and 4 were available as the minor isoform CD22alpha (deletionof amino acids 241-417). All amino acid numbers refer to the numberingof full length precursor CD22beta by Wilson, G. L. et al. (see FIG. 1 inWilson, G. L. et al., J. Exp. Med. 173:137-146 (1991)). FIG. 14 is adiagram of the deleted domains. Binding was determined by flow cytometryusing an isotype control. Binding of 10F4.4.1 was detected using goatanti-mouse IgG Alexa 488. Binding of 5E8.1.8 was detected usingbiotinylated goat anti-mouse IgG plus streptavidin PE. An adverse affecton the binding of murine 10F4.4.1 or murine 5E8.1.8 antibodies in theabsence of particular ECD domains indicated that the antibody boundthose domains. Murine 10F4.4.1 and 5E8.1.8 showed the same bindingcharacteristics under these conditions. Neither bound CD22 lackingdomain 1 or domain 2, and both bound CD22 comprising domains 1 and 2,but lacking domains 3 and 4. Using this method, it was determined that10F4.4.1 and 5E8.1.8 bind to domains 1 and 2 of human CD22, within thesequence from amino acid 22 to amino acid 240 of SEQ ID NO:27 (seeWilson, G. L. et al., (1991) supra).

Example 6 Characterization of Binding Affinity to Soluble Antigen

The binding affinity of murine and humanized 10F4 antibody for solubleCD22 extracellular domain (ECD) was determined by surface plasmonresonance measurement using a BIACORE® 3000 system (Biacore, Inc.,Piscataway, N.J.). Briefly, carboxymethylated dextran biosensor chips(CMS, Biacore Inc.) were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.These activated chips were coated with anti-CD22 IgG1 antibody 10F4(murine or humanized) by dilution to 5 μg/ml with 10 mM sodium acetate,pH 4.8, before injection at a flow rate of 5 μl/minute to achieveapproximately 500 response units (RU) of coupled antibody. Next, 1Methanolamine was injected to block unreacted groups. For kineticsmeasurements, two-fold serial dilutions of human CD22-beta-ECD-Histagged soluble antigen (approximately 500 nM to approximately 7.8 nM)were injected in PBS with 0.05% Tween 20 at 25° C. at a flow rate of 30μl/min. Association rates (k_(m)) and dissociation rates (k_(off)) werecalculated using a simple one-to-one Langmuir binding model(BIAevaluation Software version 3.2). The equilibrium dissociationconstant (Kd) was calculated as the ratio k_(off)/k_(on). Anti-CD22antibody, RFB4, was used as a control (Chemicon International, Inc.,Temecula, Calif., catalog no. CBL147). The results of this experimentare shown in Table 2 below.

TABLE 2 Anti-CD22 Binding Affinity to Soluble Human CD22 (BIACORE ®analysis) Clone k_(on)/10⁵ k_(off)/10⁻⁴ Kd (nM) Murine 10F4 0.19 2.8 15Chimeric 10F4 0.26 4.2 16 Humanized 0.18 3.5 19 10F4v1 Humanized 0.322.5 7.8 10F4v2 Control RFB4 0.33 1.4 4.2

Example 7 Characterization of Binding Affinity to Cell Surface Antigen

The binding affinity of murine 10F4.4.1 and humanized 10F4v1 and 10F4v2for human and cynomolgus monkey (cyno) CD22 expressed on the surface ofCHO cells was examined using a competition assay. Briefly, CHO cellsstably expressing full length human CD22 (SEQ ID NO:27) or cynomolgusmonkey (cyno) CD22 (SEQ ID NO:31). Anti-CD22 antibody (murine orhumanized 10F4v1 or v2) was iodinated with Iodogen® [¹²⁵I] reagent to aspecific activity of approximately 10 μci/μg. A cell-based, competitivebinding assay was performed using serially diluted, unlabeled anti-CD22antibody. Antibodies were allowed to bind to the cells for 4 hours at 4°C. Binding affinity, K_(D), of the antibodies was determined inaccordance with standard Scatchard analysis performed utilizing anon-linear curve fitting program (see, for example, Munson et al., AnalBiochem, 107: 220-239, 1980). The results of this experiment are shownin Table 3 below.

TABLE 3 10F4 MAb Binding Affinity for Human and Cyno CD22 Human CD22Cyno CD22 Antibody Kd (nM) Kd (nM) Mu 10F4.4.1 2.4 2.3 Hu 10F4v1* 1.1,1.7 1.4, 1.8 Hu 10F4v2 1.6 2.1 *repeated assays

The results indicate that murine and humanized 10F4 bind human and cynoCD22 expressed on the surface of CHO cells with approximately equivalentaffinity.

Example 8 Production of Anti-CD22 Antibody Drug Conjugates

Anti-CD22 ADCs were produced by conjugating anti-CD22 antibodies RFB4,murine 5E8, murine 10F4, humanized 10F4v1, humanized thioMAb 10F4v1(thio-10F4v1), humanized 10F4v2, and humanized 10F4v3 to the followingdrug-linker moieties: spp-DM1, smcc-DM1, MC-vc-PAB-MMAE; MC-vc-PAB-MMAF;MC-MMAE and MC-MMAF, which drug and linker moieties are disclosed hereinas well as in WO 2004/010957, published Feb. 5, 2004, and WO2006/034488,published Sep. 9, 2005 (each of which patent applications is hereinincorporated by reference in its entirety). Prior to conjugation, theantibodies were partially reduced with TCEP using standard methods inaccordance with the methodology described in WO 2004/010957. Thepartially reduced antibodies were conjugated to the above drug-linkermoieties using standard methods in accordance with the methodologydescribed in Doronina et al. (2003) Nat. Biotechnol. 21:778-784 and US2005/0238649 A1. Briefly, the partially reduced antibodies were combinedwith the drug linker moieties to allow conjugation of the moieties tocysteine residues. The conjugation reactions were quenched, and the ADCswere purified. The drug load (average number of drug moieties perantibody) for each ADC was determined by HPLC. Other useful linkers forthe preparation of ADCs include, without limitation, BMPEO, BMPS, EMCS,GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), andincluding bis-maleimide reagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO)₃,and BM(PEO)₄.

Anti-CD22 ADCs are also produced by conjugation to lysine residues ofthe antibody. Lysines of the antibody are converted to sulfhydryl groupsusing, for example, Traut's reagent (Pierce Chemical Co.) as disclosedherein. The resultant sulfhydryl groups are reactive with linkers orlinker drug molecules for the preparation of ADCs. Alternatively, ADC'sare produced by reacting a lysine on an anti-CD22 antibody with thelinker, SPP (N-succinimidyl 4-(2′-pyridyldithio)pentanoate, which may bealready attached to a drug molecule or may be subsequently reacted witha drug molecule, such as a maytansinoid. For example, an antibody ismodified by reaction with SPP, followed by conjugation with f asdisclosed in Wang, L. et al., Protein Science 14:2436-2446 (2005), whichreference is hereby incorporated by reference in its entirety. Lysineresidues on an anti-CD22 antibody may also be reacted with the linker,SMCC (Pierce Chemical Co.), at pH 7-9 such that the amine-reactiveN-hydroxysuccinimide (NHS ester) of SMCC forms a stable amide bond withthe antibody. The sulfhydryl-reactive maleimide group of SMCC is reactedwith the sulfhydryl group of DM1 at pH 6.5-7.5 (see Pierce Chemical Co.,piercenet.com) to form the ADC. Lysine or cysteine residues are reactedwith linker-drug to produce ADCs comprising an average drug load ofapproximately 1-8 linker drug molecules per antibody, alternatively1-6,1-4, 1-3 or 1-2 linker drug molecules per antibody.

ADCs anti-CD22(RFB4)-SMCC-DM1 and anti-GP120-SMCC-DM1 were preparedaccording to this method, where RFB4-smcc-DM1 was prepared at low(1.95), medium (3.7) and high (6.75) drug loads. Anti-GP120-smcc-DM1 wasprepared at high (6.1) drug load. These ADCs were shown to beefficacious in vivo, as shown in Example 9 and Table 9, herein below.

Example 9 Efficacy of Anti-CD22 Antibody Drug Conjugates

In Vitro Studies of Efficacy Determinants.

The determinants of anti-CD22 ADC (or TDC) efficacy in a lymphoma cellline were determined. It is known that CD22 expressed on the surface ofB cells is internalized upon binding of its ligand(s) or antibodies(Sato, S. et al., Immunity 5:551-562 (1996)). To test whether and howthe level of B cell surface expression of CD22 and/or internalization ofCD22 affect efficacy, the following in vitro studies were performed.

Surface expression of human CD22 on multiple lymphoma cell lines.Nineteen lymphoma cell lines expressing varying amounts of CD22 on theirsurface were cultured and harvested in log phase growth. Cells wereresuspended in FACS wash buffer (PBS; 0.5% bovine serum albumin; 0.1%sodium azide) containing 100 μg/ml each normal mouse IgG and normalhuman IgG and maintained on ice. Approximately 1×10̂6 cells/100 μl werestained with anti-huCD22 APC (mIgG1, clone RFB4, Southern Biotech#9361-11) or murine IgG1 APC isotype (BD Pharmingen #555751) for 30minutes on ice. Dead cells were stained with 7-AAD (BD Pharmingen#559925). Data were acquired on a BD FacsCalibur™ flow cytometer andanalyzed with FlowJo™ software. The IC50 determination forhu10F4v3-SMCC-DM1 or each free drug (DM1, MMAF, or MMAE) were determinedby culturing lymphoma cells as above, harvesting the cultured cells inlog phase and seeding 5,000 cells in 90 μl culture medium per well in 96well plate. ADC and free drug were diluted serially within the detectionrange (starting at 300 μg/ml for ADC, or 90 nM for free drug anddiluting to essentially zero assay target). Aliquots of 10 μl dilutedADC or free drug were added to replicate wells containing cells andincubated for 3 days at 37° C. To each well, 100 μl CellTiter Glo™ wasadded and incubated for 30 min. Chemiluminescence was detected and datawere analyzed using Prism™ software. The results are shown in FIG. 6A,in which high surface CD22 levels correlate with low IC50 (higherefficacy) of hu10F4v3-SMCC-DM1. FIG. 6C indicates that a strongercorrelation exists between the intrinsic sensitivity of the cells tofree drug and the IC50 of the ADC.

Internalization of hu10F4v3-SMCC-DM1 was Determined by FACS Assay.

Briefly, lymphoma cells were stained by standard FACS techniques withCD22-FITC(RFB4) in the presence of hu10F4v3-SMCC-DM1 and incubated onice for 20-30 minutes. To determine CD22 levels on the cell surfaceafter the initial staining, cells were washed in cold RPMI/10% FBS mediaand 200 μl pre-warmed RPMI/10% FBS was added and incubated at 37° C. for15 minutes. 80 μl staining buffer and 20 μl heat-inactivated normalmouse serum (HI NMS) were added, followed by incubation on ice for 15minutes. Anti-DM1-Alexa-647 was added, incubated on ice for 20-30minutes and cells were washed and fixed with 200 μl PBS/1%paraformaldehyde prior to FACS analysis. To determine surface andinternal staining of CD22 after the initial staining, cells were washedwith cold RPMI/10% FBS, pre-warmed RPMI/10% FBS was added and the cellsincubated for 15 minutes at 37° C. Cells were then washed with FACS Washand fixed with Fix Reagent A (Dako™ #k2311) at room temperature for 15minutes, and the step was repeated with Fix Reagent B (Dako™). Stainingbuffer and HI NMS were added and the cell mixture was incubated on icefor 15 min. Fix Reagent B was added, followed by anti-DM1-Alexa-647 andincubated at room temperature for 20-30 minutes. Cells were washed inFACS Wash and fixed in PBS/1% paraformaldehyde. FACS analysis wasperformed on each cell mixture (surface, surface post-internalization,and internal staining) using a BD FacsCalibur™ flow cytometer andanalyzed with FlowJo™ software. The results are shown in FIG. 6B inwhich high amounts of internalized DM1 correlated with low IC50 (highefficacy); and in FIG. 6D in which internalized DM1 is visualized byfluorescent microscopy.

In Vivo Efficacy Studies.

To test the efficacy of toxin-conjugated or unconjugated anti-CD22monoclonal antibodies for the ability to reduce tumor volume in vivo,the following protocol was employed.

SCID mice were each inoculated subcutaneously in the flank with 2×10⁷ ahuman B-cell lymphoma cell line. The human cell lines included humanBurkitt lymphoma cell lines Daudi, Ramos, and Raji cells (available fromthe American Type Culture Collection, Manassas, Va., USA), and otherB-cell lines including U-698-M cells and Su-DHL-4 cells (available fromDSMZ, Braunschweig, Germany; Su-DHL-4 cells were transfected with theluciferase reporter gene), DoHH2 cells (Kluin-Neilemans, H. C. (1991),supra), and Granta-519 (Mantle cell lymphoma cells, Jadayel, D. M. etal., Leukemia 11(1):64-72 (1997)), and BJAB-luc cells (BJAB human B-celllymphoblastoid cell line which expresses reporter gene luciferase. Whenthe tumors reached a mean tumor volume of between 100-200 mm3, the micewere divided into groups, and treated on day 0 by intravenous injectionwith toxin-conjugated antibody or unconjugated antibody as shown inTables 4-16, below.

Anti-CD22 Maytansine Drug Conjugates Reduce B-Cell Tumor Volume

Sixty-five SCID mice were injected with 2×10̂7 BJAB-luc cellssubcutaneously in a volume of 0.2 ml per mouse in the flank. Cells weresuspended in HBSS. When the mean tumor size reached 100-200 mm̂3, micewere randomly grouped into four groups of 9 mice each and given a singleI.V. treatment (via the tail vein) of the anti-CD22 or control antibodyindicated in Table 4, below.

TABLE 4 In Vivo Tumor Volume Reduction Antibody Administration Drugratio Antibody Dose Ab Dose DM1 (Drug administered TI PR CR (mg/kg)(μg/m²) moieties/Ab) anti-Her2- 9/9 0 0 4.2 200 3.2 smcc-DM1 mu10F4- 9/92 0 3.0 200 4.6 smcc-DM1 hu10F4v2- 9/9 0 0 3.4 200 4.0 smcc-DM1 hu10F4v19/9 0 0 3.4 — — “TI”—tumor incidence at the last time point of eachgroup; the numerator refers to the number of tumor-bearing animals andthe denominator refers to total number of animals. “PR” refers to thenumber of animals with tumor regressed 50-99% from its initial volume.“CR” refers to the number of animals attaining complete remission.

Mean tumor volume was monitored in each treatment group for 32 dayspost-antibody injection. Tumor measurements were taken with calipers.Efficacy of the toxin-conjugated anti-CD22 antibodies was determined bycomparison to the control and unconjugated antibodies. The results areshown in FIG. 7A. The murine and humanized 10F4v1-smcc-DM1 monoclonalantibodies significantly slowed tumor growth relative to unconjugatedanti-CD22 antibody and non-specific control antibody.

Using the same protocol as above, an assay was performed comparingtoxin-conjugated humanized 10F4v2 to toxin-conjugated murine and nakedhumanized antibody as indicated in Table 5, below.

TABLE 5 In Vivo Tumor Volume Reduction Antibody Administration Drugratio Antibody Dose Ab Dose DM1 (Drug administered TI PR CR (mg/kg)(μg/m²) moieties/Ab) anti-Her2- 9/9 0 0 4.2 200 3.2 smcc-DM1 mu10F4- 7/91 2 4.7 200 2.9 smcc-DM1 hu10F4v2- 8/9 1 1 4.5 200 3.0 smcc-DM1 hu10F4v29/9 0 0 4.5 — — “TI”—tumor incidence at the last time point of eachgroup; the numerator refers to the number of tumor-animals in the studygroup and the denominator refers to total number of animals. “PR” refersto the number of animals with tumor regressed 50-99% from its initialvolume. “CR” refers to the number of animals attaining completeremission.

Mean tumor volume was monitored in each treatment group for 32 dayspost-antibody injection. Tumor measurements were taken with calipers.Efficacy of the toxin-conjugated anti-CD22 antibodies was determined bycomparison to the control and unconjugated antibodies. The results areshown in FIG. 7B. The murine 10F4-smcc-DM1 and humanized 10F4v2-smcc-DM1monoclonal antibodies significantly slowed tumor growth relative tounconjugated anti-CD22 antibody and non-specific control antibody.

Anti-CD22 antibody was conjugated to DM1 via the spp linker or the smcclinker according to conjugation methods disclosed herein. The nakedanti-CD20 antibody was used as a positive control and the toxinconjugates, anti-HER2-spp-DM1 and anti-HER2 were used as negativecontrols. Eighty SCID mice were injected with 2×10̂7 BJAB-luc cellssubcutaneously in a volume of 0.2 ml per mouse in the flank. Cells weresuspended in HBSS. When the mean tumor size reached 100-200 mm̂3, themice were randomly grouped into six groups of 10 mice each andintravenous injection of test or control antibodies was performed. Doseswere repeated once each week for a total of three doses. See Table 6.

TABLE 6 In Vivo Tumor Volume Reduction Antibody Administration AntibodyDose Ab Dose DM1 administered (mg/kg) (μg/m²) anti-Her2- 4 214 spp-DM1*anti-Her2- 6.9 405 smcc-DM1** anti-CD22- 5 214 spp-DM1* anti-CD22- 2.5107 spp-DM1 anti-CD22- 10 405 smcc-DM1** naked anti- 10 — CD22 *Matcheddrug load **Matched drug load

Mean tumor volume was monitored twice each week for 3 weeks and thenonce each week thereafter for a total of 8 weeks. Changes in tumorvolume over time (FIG. 7C) show that the anti-CD22-spp-DM1 dosed at 214and 107 μg/m̂2 DM1 and anti-CD22 dosed at 405 μg/m̂2 showed robust andcomparable anti-tumor activity in BJAB-luc xenograft tumors. Allanti-CD22 ADC groups showed complete responses.

The anti-CD22 antibodies, RFB4, 5E8, and 7A2 were conjugated to DM1 viathe smcc linker according to conjugation methods disclosed herein. Thetoxin conjugate, anti-HER 2-smcc-DM 1 (referred to interchangeablyherein as HER-smcc-DM 1 or HER2-smcc-DM1), was used as negative control.

The ability of these antibodies to reduce tumor volume in variousxenografts in SCID mice was examined. The human B-cell lymphoma celllines used to generate xenograft tumors in mice were Ramos cells andBJAB-luc cells. For each xenograft, SCID mice were injected with 5×10̂6human B-cell lymphoma Ramos cells subcutaneously in a volume of 0.1 mlper mouse in the flank (or 2×10̂7 BJAB-luc cells in 0.2 ml). Cells weresuspended in HBSS. When the mean tumor size reached 100-200 mm̂3, themice were randomly grouped into groups of 8-10 mice each, and each mousewas given a single intravenous injection of test or control antibody.DM1 drug loading was normalized to 200 μg/m² for each group to providethe dose of DM1 administered. Mean tumor volume was monitored twice eachweek for 4 weeks. The results are shown below in Tables 7 and 8 andplotted in FIGS. 8A and 8B, respectively.

TABLE 7 In Vivo Tumor Volume Reduction, Ramos Xenograft AntibodyAdministration Antibody Dose Ab Dose DM1 administered (mg/kg) (μg/m²)anti-HER2- 4.2 200 smcc-DM1 Anti- 3.8 200 CD22(7A2)- smcc-DM1 anti- 3.8200 CD22(5E8)- smcc-DM1 Anti- 3.2 200 CD22(RFB4)- smcc-DM1

TABLE 8 In Vivo Tumor Volume Reduction, BJAB-luc Xenograft AntibodyAdministration Drug ratio Antibody Dose Ab Dose DM1 (Drug administered(mg/kg) (μg/m²) moieties/Ab) anti-HER2- 4.2 200 3.2 smcc-DM1 Anti-CD223.8 200 3.6 (7A2)-smcc- DM1 Anti- 3.8 200 3.6 CD22(5E8)- smcc-DM1 Anti-3.2 200 4.25 CD22(RFB4)- smcc-DM1

These results show that anti-CD22-smcc-DM1 antibody drug conjugatessignificantly reduce B-cell tumor volume in Ramos, and BJAB-lucxenografts relative to control antibody or naked anti-CD22 antibody.

The affect of antibody drug load (average number of drug moleculesconjugated per antibody in a population of antibodies) on the ability ofanti-CD22-smcc-DM 1 antibody drug conjugates to reduce tumor volume inBJAB-luc SCID mouse xenografts was examined. One hundred forty SCID micewere injected with 2×10̂7 BJAB-luc cells subcutaneously in a volume of0.2 ml per mouse in the flank. Cells were suspended in HBSS. When themean tumor size reached 100-200 mm̂3, the mice were randomly grouped intogroups of 8-10 mice each, and each mouse was given a single intravenousinjection of test or control antibody. Populations ofanti-CD22(RFB4)-smcc-DM1 having relative low, medium or high drug loads(average drug loads 1.95, 3.7, or 6.75 conjugated DM1 molecules perantibody, respectively) were administered as the test antibodies. NakedRFB4 antibody and anti-GP120-smcc-DM1 (high drug load) were thecontrols. The doses of antibody drug conjugates (test and control) werenormalized to a dose of 5 mg/kg protein level. Linker conjugateattachment to the antibodies was via lysine residues. See Table 9.

TABLE 9 In Vivo Tumor Volume Reduction, BJAB-luc XenograftAnti-CD22(RFB4)-smcc-DM1 Administration Drug ratio (Drug Antibody DoseAb Dose DM1 moieties/ administered (mg/kg) (μg/m²) Ab) anti-CD22(RFB4)10 — — (naked antibody) anti-CD22(RFB4)- 5 144 1.95 smcc-DM1 (low)anti-CD22(RFB4)- 5 273 3.7 smcc-DM1 (medium) anti-CD22(RFB4)- 5 497 6.75smcc-DM1 (high) anti-GP120-smcc- 5 449 6.1 DM1 (high)

When dosed at a matching protein level (5 mg/kg),anti-CD22(RFB4)-smcc-DM1 loaded with a high drug load (6.75 DM1molecules per antibody molecule) reduced tumor volume slightly more thanthe antibody drug conjugate with a medium load of 3.7, whereas theaffects of the antibody drug conjugate with a low drug load was notdifferent from control conjugate or naked antibody. The results areplotted in FIG. 9.

Anti-CD22 Auristatin Drug Conjugates Reduce B-Cell Tumor Volume

The affect of anti-CD22 auristatin MMAF drug conjugates on tumor volumein mouse xenografts was examined. Anti-CD22(RFB4) and control antibodyanti-GP120 were conjugated to MMAF via a MC-vcPAB linker or a MC linkeraccording to methods disclosed herein. SCID mice were injected with5×10̂6 Ramos cells subcutaneously in a volume of 0.2 ml per mouse in theflank. Cells were suspended in HBSS. When the mean tumor size reached100-200 mm̂3, the mice were randomly grouped into groups of 8-10 miceeach, and each mouse was given a single intravenous injection of test orcontrol antibody. Drug dose, drug load (drug ratio) and antibody doseadministered to the mice are shown in Table 10.

TABLE 10 In Vivo Tumor Volume Reduction, Ramos Xenograft Anti-CD22(RFB4)MMAF Conjugate Administration Dose Drug ratio Antibody MMAF Dose Ab(Drug administered (μg/m²) (mg/kg) moieties/Ab) anti-CD22(RFB4)- 405 6.64.2 MCvcPAB-MMAF anti-CD22(RFB4)- 405 6.9 4.0 MC-MMAF anti-GP120- 4055.8 4.8 MCvcPAB-MMAF anti-GP120-MC- 405 5.9 4.7 MMAF

Anti-CD22-MC-MMAF showed comparable activity compared toanti-CD22-MC-vc-PAB-MMAF in Ramos RA1 xenografts. The results areplotted in FIG. 10.

The affect of anti-CD22 auristatin MMAE and DM1 drug conjugates on tumorvolume in mouse xenografts was examined. Anti-CD22(RFB4) and controlantibody anti-GP 120 were conjugated to MMAE via a MC-vcPAB linker or aMC linker or to DM1 via a smcc linker according to methods disclosedherein. SCID mice were injected with 5×10̂6 Ramos cells subcutaneously ina volume of 0.1 ml per mouse in the flank. Cells were suspended in HBSS.PBS was administered as a control. When the mean tumor size reached100-200 mm̂3, the mice were randomly grouped into groups of 8-10 miceeach, and each mouse was given a single intravenous injection of test orcontrol antibody. Drug dose, drug load (drug ratio) and antibody doseadministered to the mice are shown in Table 11.

TABLE 11 In Vivo Tumor Volume Reduction, Ramos Xenograft Anti-CD22(RFB4)MMAE and DM1 Conjugate Administration Dose MMAE Drug ratio Antibody orDM1 Dose Ab (Drug administered (μg/m²) (mg/kg) moieties/Ab)Anti-GP120-smcc- 405 6.7 4.1 DM1 Anti-CD22(RFB4)- 405 6.5 4.25 smcc-DM1Anti-GP120- 405 6.0 4.7 MCvcPAB-MMAE antiCD22(RFB4)- 405 6.3 4.5MCvcPAB-MMAE PBS — — —

Anti-CD22-MCvcPAB-MMAE showed potent anti-tumor activity in Ramos RA1xenografts. The anti-CD22-MCvcPAB-MMAE showed superior activity comparedto antiCD22-smcc-DM1. The ADC control, anti-GP120-MCvcPAB-MMAE, did notshow significant activity. The results are plotted in FIG. 11.

The affect of anti-CD22 auristatin MMAF and DM1 drug conjugates on tumorvolume in mouse xenografts was examined. Anti-CD22 hu10F4v2-MC-MMAF,hu10F4v2 and thio-10F4v1-MC-MMAF were administered and compared foraffect on tumor volume. Control antibodies were anti-Her2-MC-MMAF andanti-Her2-smcc-DM1. SCID mice were injected with 2×10̂7 BJAB-luc cellssubcutaneously in a volume of 0.2 ml per mouse in the flank. Cells weresuspended in HBSS. When the mean tumor size reached 100-200 mm̂3, themice were randomly grouped into groups of 8-10 mice each, and each mousewas given a single intravenous injection of test or control antibody.“Thio” refers to a thioMab, as disclosed herein, in which thelinker-drug moiety is conjugated to the antibody via a cysteineengineered site on the antibody. Drug dose, drug load (drug ratio) andantibody dose administered to the mice are shown in Table 12.

TABLE 12 In Vivo Tumor Volume Reduction, BJAB-luc Xenograft Hu10F4 MMAFand DM1 Conjugate Administration Dose MMAF Drug ratio Antibody or DM1Dose Ab (Drug administered (μg/m²) (mg/kg) moieties/Ab) Anti-Her2-MC-100 1.1 6.3 MMAF Hu10F4v2-MC- 100 2.0 3.4 MMAF Hu10F4v2-MC- 50 1.0 3.4MMAF Thio-hu10F4v1-MC- 100 4.6 1.5 MMAF Thio-hu10F4v1-MC- 50 2.3 1.5MMAF Anti-Her2-smcc-DM1 200 4.2 3.2 Hu10F4v2-smcc- 200 4.5 3.0 DM1Hu10F4v2-smcc- 100 2.3 3.0 DM1

Hu10F4v2 ADCs showed potent anti-tumor activity in BJAB-luc xenografts.The results are plotted in FIG. 12.

Using procedures as disclosed in the above experiments,hu10F4v3-smcc-DM1 and -MC-MMAF ADC efficacy in different xenografts atdifferent doses was examined. Xenografts of SuDHL4-luc, DoHH2, andGranta-519 xenografts were prepared as disclosed herein, above. When thetumor size reached 100-200 mm̂3, the mice were randomly grouped intogroups of 8-10 mice each, and each mouse was given a single intravenousinjection of test or control antibody. Drug dose, drug load (drug ratio)and antibody dose administered to the mice are shown in Tables 13A-13Cand the results are shown in FIGS. 13A-13C.

TABLE 13A In Vivo Tumor Volume Reduction, Hu10F4v3 MMAF and DM1Conjugate Administration In SuDHL-4-luc Xenografts Dose MMAF or AntibodyDM1 Dose Ab Drug ratio administered (μg/m²) (mg/kg) (Drug/Ab)Anti-Her2-smcc- 600 11.9 3.3 DM1 Hu10F4v3-smcc- 600 13.6 2.9 DM1Hu10F4v3-smcc- 300 6.8 2.9 DM1 Anti-Her2-MC- 600 9.9 4.0 MMAFHu10F4v3-MC- 600 13.3 3.0 MMAF Hu10F4v3-MC- 300 6.6 3.0 MMAF

TABLE 13B In Vivo Tumor Volume Reduction, Hu10F4v3 MMAF and DM1Conjugate Administration In DoHH2 Xenografts Dose MMAF or Antibody DM1Dose Ab Drug ratio administered (μg/m²) (mg/kg) (Drug/Ab)Anti-Her2-smcc- 600 11.9 3.3 DM1 Hu10F4v3-smcc- 600 11.8 3.35 DM1Hu10F4v3-smcc- 300 5.9 3.35 DM1 Anti-Her2-MC- 600 9.9 4.0 MMAFHu10F4v3-MC- 600 13.1 3.04 MMAF Hu10F4v3-MC- 300 6.6 3.04 MMAF Nakedhu10F4v3 — 13.1 —

TABLE 13C In Vivo Tumor Volume Reduction, Hu10F4v3 MMAF and DM1Conjugate Administration In Granta-519 Xenografts Dose MMAF or AntibodyDM1 Dose Ab Drug ratio administered (μg/m²) (mg/kg) (Drug/Ab)Anti-Her2-smcc- 300 5.9 3.3 DM1 Hu10F4v3-smcc- 300 5.9 3.35 DM1Hu10F4v3-smcc- 150 2.9 3.35 DM1 Anti-Her2-MC- 300 4.9 4.0 MMAFHu10F4v3-MC- 300 6.6 3.04 MMAF Hu10F4v3-MC- 150 3.3 3.04 MMAF Nakedhu10F4v3 — 6.6 —

Anti-CD22 hu10F4v3-smcc-DM1 and -MC-MMAF ADCs showed potent tumorreduction in all of the xenograft models tested.

Example 10 Preparation of Cysteine Engineered Anti-CD22 Antibodies

Preparation of cysteine engineered anti-CD22 antibodies was performed asdisclosed herein. DNA encoding the 10F4v3 antibody, having the samevariable and constant region sequences as 10F4v2 (light chain, SEQ IDNO:87; and heavy chain, SEQ ID NO:88, FIG. 5B), was mutagenized bymethods disclosed herein to modify the light chain, the heavy chain orthe Fc region of the heavy chain. DNA encoding the light chain wasmutagenized to substitute cysteine for valine at Kabat position 205 inthe light chain (sequential position 210) as shown in FIG. 17A (lightchain SEQ ID NO:91 of humanized antibody 10F4v3 thiomab). DNA encodingthe heavy chain was mutagenized to substitute cysteine for alanine at EUposition 118 in the heavy chain (sequential position 121) as shown inFIG. 17B (heavy chain SEQ ID NO:92 of humanized antibody 10F4v3thiomab). The Fc region was mutagenized to substitute cysteine forserine at EU position 400 in the heavy chain Fc region (sequentialposition 403) as shown in FIG. 17C (heavy chain SEQ ID NO:93).

Preparation of Cysteine Engineered Anti-CD22 Antibodies for Conjugationby Reduction and Reoxidation

Full length, cysteine engineered anti-CD22 monoclonal antibodies(ThioMabs) expressed in CHO cells are dissolved in 500 mM sodium borateand 500 mM sodium chloride at about pH 8.0 and reduced with about a50-100 fold excess of 1 mM TCEP (tris(2-carboxyethyl)phosphinehydrochloride; Getz et al (1999) Anal. Biochem. Vol 273:73-80; SoltecVentures, Beverly, Mass.) for about 1-2 hrs at 37° C. The reducedThioMab is diluted and loaded onto a HiTrap S column in 10 mM sodiumacetate, pH 5, and eluted with PBS containing 0.3M sodium chloride. Theeluted reduced ThioMab is treated with 2 mM dehydroascorbic acid (dhAA)at pH 7 for 3 hours, or 2 mM aqueous copper sulfate (CuSO₄) at roomtemperature overnight. Ambient air oxidation may also be effective. Thebuffer is exchanged by elution over Sephadex G25 resin and eluted withPBS with 1 mM DTPA. The thiol/Ab value is checked by determining thereduced antibody concentration from the absorbance at 280 nm of thesolution and the thiol concentration by reaction with DTNB (Aldrich,Milwaukee, Wis.) and determination of the absorbance at 412 nm.

Example 11 Preparation of Cysteine Engineered Anti-CD22 Antibody DrugConjugates by Conjugation of Cysteine Engineered Anti-CD22 Antibodiesand Drug-Linker Intermediates

After the reduction and reoxidation procedures of Example 10, thecysteine engineered anti-CD22 antibody is dissolved in PBS (phosphatebuffered saline) buffer and chilled on ice. About 1.5 molar equivalentsrelative to engineered cysteines per antibody of an auristatin druglinker intermediate, such as MC-MMAE (maleimidocaproyl-monomethylauristatin E), MC-MMAF, MC-val-cit-PAB-MMAE, or MC-val-cit-PAB-MMAF,with a thiol-reactive functional group such as maleimido, is dissolvedin DMSO, diluted in acetonitrile and water, and added to the chilledreduced, reoxidized antibody in PBS. After about one hour, an excess ofmaleimide is added to quench the reaction and cap any unreacted antibodythiol groups. The reaction mixture is concentrated by centrifugalultrafiltration and the cysteine engineered anti-CD22 antibody drugconjugate is purified and desalted by elution through G25 resin in PBS,filtered through 0.2 μm filters under sterile conditions, and frozen forstorage.

Preparation of hu 10F4v3 HC(A118C) thiomab-BMPEO-DM1 was performed asfollows. The free cysteine on hu 10F4v3 HC(A118C) thiomab was modifiedby the bis-maleimido reagent BM(PEO)₄ (Pierce Chemical), leaving anunreacted maleimido group on the surface of the antibody. This wasaccomplished by dissolving BM(PEO)4 in a 50% ethanol/water mixture to aconcentration of 10 mM and adding a tenfold molar excess of BM(PEO)₄ toa solution containing hu4D5Fabv8-(V110C) ThioFab in phosphate bufferedsaline at a concentration of approximately 1.6 mg/ml (10 micromolar) andallowing it to react for 1 hour. Excess BM(PEO)4 was removed by gelfiltration (HiTrap column, Pharmacia) in 30 mM citrate, pH 6 with 150 mMNaCl buffer. An approximate 10 fold molar excess DM1 dissolved indimethyl acetamide (DMA) was added to the hu4D5Fabv8 ThioFab-BMPEOintermediate. Dimethylformamide (DMF) may also be employed to dissolvethe drug moiety reagent. The reaction mixture was allowed to reactovernight before gel filtration or dialysis into PBS to remove unreacteddrug. Gel filtration on S200 columns in PBS was used to remove highmolecular weight aggregates and furnish purified hu 10F4v3 HC(A118C)thiomab-BMPEO-DM1.

By the same protocols, control HC (A118C) MAb-MC-MMAF, control HCThioMAb-MC-MMAF, control HCThioMAb-MCvcPAB-MMAE, and control HCThioMab-BMPEO-DM1 were prepared.

By the procedures above, the following cysteine engineered anti-CD22antibody drug conjugates were prepared and tested:

thio hu thio-HC-10F4v3-MC-MMAF by conjugation of A118C thio hu 10F4v3and MC-MMAF;

thio hu thio-HC-10F4v3-MC-val-cit-PAB-MMAE by conjugation of A118C thiohu 10F4v3 and MC-val-cit-PAB-MMAE;

thio hu HC-10F4v3-bmpeo-DM1 by conjugation of A118C thio hu HC-10F4v3and bmpeo-DM 1;

thio hu LC-10F4v3-MC-val-cit-PAB-MMAE by conjugation of V205C thio huLC-10F4v3 and MC-val-cit-PAB-MMAE; and

thio hu Fc-10F4v3-MC-val-cit-PAB-MMAE by conjugation of S400C thio huFc-10F4v3 and MC-val-cit-PAB-MMAE.

Example 12 Characterization of Binding Affinity of Cysteine EngineeredThioMAb Drug Conjugates to Cell Surface Antigen

The binding affinity of thio hu 10F4v3 drug conjugates to CD22 expressedon BJAB-lucs cells was determined by FACS analysis. Briefly,approximately 1×10̂6 cells in 100 μl were contacted with varying amountsof one of the following anti-CD22 ThioMAb drug conjugates: thio huLC(V205C) 10F4v3-MCvcPAB-MMAE, thio hu Fc(S400C) 10F4v3-MCvcPAB-MMAE,thio hu HC(A118C) 10F4v3-MCvcPAB-MMAE, thio hu HC(A118C) 10F4v3-MC-MMAF,or thio hu HC(A118C) 10F4v3-BMPEO-DM1 (see FIGS. 18A-18E, respectively).Anti-CD22 antibody bound to the cell surface was detected usingbiotinylated goat anti-huFc plus Streptavidin-PE. The plots of FIGS.18A-18E indicate that antigen binding was approximately the same for allof the thiomab drug conjugates tested.

Example 13 Assay for In Vivo Tumor Volume Reduction by Anti-CD22 ThioMabDrug Conjugates

The ability of the thiomab drug conjugates prepared according to Example11 to reduce B-cell tumor volume in xenograft models was testedaccording to the procedure disclosed in Example 9, herein. To SCID micehaving Granta-519 cell xenograft tumors, control and anti-CD22 humanized10F4v3 thiomab drug conjugates were administered at Day 0 in the dosesshown in Table 14, below. The control HC(A118C) thiomab was anti-HER24D5 antibody.

TABLE 14 In Vivo Tumor Volume Reduction, Thio Hu10F4v3 MMAE and MMAFConjugate Administration In Granta-519 Xenografts Dose MMAF or DM1 DoseAb Drug ratio Antibody administered (μg/m²) (mg/kg) (Drug/Ab) ThioControl HC(A118C)-MC-MMAF 100 3.99 1.65 Thio Control HC(A118C)-MCvcPAB-100 4.33 1.55 MMAE Thio 10F4v3-HC(A118C)-MC-MMAF 100 3.41 1.95 Thio10F4v3-LC(V205C)-MCvcPAB- 100 4.23 1.6 MMAE Thio10F4v3-HC(A118C)-MCvcPAB- 100 3.76 1.8 MMAE Thio10F4v3-Fc(S400C)-MCvcPAB- 100 4.23 1.6 MMAE

The results of this experiment are shown in FIG. 19. Administration ofthe thio 10F4v3-LC-(V205C)-MCvcPAB-MMAE and thio10F4v3-HC(A118C)-MCvcPAB-MMAE thiomab drug conjugates at the doses shownin Table 14 caused a reduction in mean tumor volume for the duration ofthe study.

Additional thiomab drug conjugates were tested in Granta-519 xenograftsin CB17 SCID mice using the same protocol, although different drug doseswere tested. The control antibody or control thiomab was anti-HER24D5antibody or HC(A118C) thiomab. The results are shown in Table 15, below.

TABLE 15 In Vivo Tumor Volume Reduction, Thio Hu10F4v3 MMAE, MMAF, andDM1 Conjugate Administration In Granta-519 Xenografts Dose MMAF Dose orDM1 Ab Drug ratio Antibody administered (μg/m²) (mg/kg) (Drug/Ab)10F4v3-MC-MMAF 150 3.2 3.1 Thio Control HC(A118C)-BMPEO-DM1 300 10.3 1.9Thio 10F4v3-HC(A118C)-BMPEO-DM1 150 5.2 1.9 Thio10F4v3-HC(A118C)-BMPEO-DM1 300 10.4 1.9 Thio Control HC(A118C)-MCvcPAB-150 6.5 1.55 MMAE Thio 10F4v3-HC(A118C)-MCvcPAB- 150 5.3 1.9 MMAE Thio10F4v3-HC(A118C)-MCvcPAB- 75 2.7 1.9 MMAE Thio Control HC(A118C)-MC-MMAF150 5.2 1.9 Thio 10F4v3-HC(A118C)-MC-MMAF 150 5.1 1.95 Thio10F4v3-HC(A118C)-MC-MMAF 75 2.6 1.95

The results of this experiment are shown in FIG. 20A. Administration ofthe thio 10F4v3-HC(A118C)-MCvcPAB-MMAE thiomab drug conjugate at 150 and75 μg/m² caused a reduction in mean tumor volume for the duration of thestudy. In the same study, the percent body weight change in the first 7days was determined in each dosage group. The results plotted in FIG.20B indicate administration of these thiomab drug conjugates did notcause weight loss during this time.

In a similar study, using the same xenograft study protocol as disclosedin the above examples, varying the TDCs and doses administered, theefficacy of TDCs in follicular lymphoma DOHH2 xenografts in CB17 SCIDmice was studied. The TDCs and doses are shown in Table 16, below.

TABLE 16 In Vivo Tumor Volume Reduction, Thio Hu10F4v3 MMAE, MMAF, andDM1 Conjugate Administration In DOHH2 Xenografts Dose MMAF Dose or DM1Ab Drug ratio Antibody administered (μg/m²) (mg/kg) (Drug/Ab)10F4v3-MC-MMAF 300 6.4 3.1 Thio Control HC(A118C)-BMPEO-DM1 600 21.91.79 Thio 10F4v3-HC(A118C)-BMPEO-DM1 600 20.8 1.9 Thio10F4v3-HC(A118C)-BMPEO-DM1 300 10.4 1.9 Thio Control HC(A118C)-MCvcPAB-600 26.0 1.55 MMAE Thio 10F4v3-HC(A118C)-MCvcPAB- 600 21.4 1.9 MMAE Thio10F4v3-HC(A118C)-MCvcPAB- 300 10.7 1.9 MMAE Thio ControlHC(A118C)-MC-MMAF 600 20.8 1.9 Thio 10F4v3-HC(A118C)-MC-MMAF 600 20.41.95 Thio 10F4v3-HC(A118C)-MC-MMAF 300 10.2 1.95

FIG. 20C is a graph plotting changes in mean tumor volume over time inthe follicular lymphoma DOHH2 xenograft in CB17 SCID mice treated withthe same heavy chain A118C anti-CD22 TDCs, but at higher doses as shownin Table 16. The anti-CD22 10F4v3-HC(A118C)-MCvcPAB-MMAE TDC appeared tobe the most efficacious of the test agents in this study. However, atthe increased dose levels in this experiment, some efficacy was noted inthe anti-HER2-HC(A118C)-MCvcPAB-MMAE controls. This activity is possiblyattributable to release of the drug from the ADC in circulation. Theanti-CD22 hu10F4-HC(A118C)-MC-MMAF and -BMPEO-DM1 test agents showedintermediate efficacy and, consistent with the increased stability ofthese linkers, the non-binding anti-HER2 controls showed littleactivity. FIG. 20D is a plot of percent weight change in the mice fromthe DOHH2 xenograft study showing that there was no significant changein weight during the first 14 days of the study.

Example 14 Safety of Anti-CD22 Drug Conjugates in Rats and CynomolgusMonkeys

The hu10F4 anti-CD22 antibody cross-reacts with cynomolgus (cyno) monkeyCD22 with an affinity equivalent to human CD22. The hu10F4 anti-CD22antibody does not cross-react with rat CD22. As a result, thetarget-independent and target-dependent safety and toxicity of anti-CD22drug conjugates were assessed in rat and cyno, respectively.

Safety and Toxicity in Rats.

For safety and toxicity studies in rats, two studies were performed. Inone study, rats were dosed intravenously on Day 1 withhu10F4v3-SMCC-DM1, —SPP-DM1, -MC-vc-PAB-MMAE, or -MC-MMAF conjugates inwhich the drug was linked via a cleavable (-vc- or -spp-) or uncleavable(MC or SMCC (also referred to as MCC)) linker. Vehicle was administeredas the control. Blood samples were collected on Day 5 forpharmacokinetic analysis, and on Day 12 (at necropsy). Clinicalobservations and body weight recordings were conducted at least threetimes per week. Serum AST (aspartate aminotransferase) was monitored asan indication of toxicity. Serum AST levels were increased at Day 5relative to Day 0 in rats dosed with 20 mg/kg hu10F4v3-vcMMAE andhu10F4v3-SPP-DM1 comprising cleavable linkers (FIG. 21A). Neutrophillevels were increased at Day 5 relative to Day 0 in rats dosed with 20mg/kg hu10F4v3-MC-MMAF or hu10F4v3-MCC-DM1 (uncleavable linkers, FIG.21B). Neutrophil levels were decreased at Day 5 relative to Day 0 inrats dosed with hu10F4v3-vc-MMAE or hu10F4v3-SPP-DM1. Increased serumAST and decreased neutrophils in rats dosed with ADCs comprisingcleavable linkers indicates increased toxicity of such ADCs

In the same rat study, six animals per group were dosed with 20, 40, or60 mg/kg hu10F4v3-MC-MMAF or hu10F4v3-SMCC-DM1 at Day 1 and monitoredfor twelve days. In animals dosed with hu10F4v3-MC-MMAF, there were noobservations in the following indicators: decreased body weight,increases in serum liver enzymes, decreases in platelets, or decreasesin neutrophils. In rats dosed with hu10F4v3-SMCC-DM1, reversibledecreased body weight and reversible increases in serum liver enzymeswere observed at dose levels of 40 and 60 mg/kg, whereas reversibledecreases in neutrophils and transient decreases in platelets wereobserved at 60 mg/kg doses.

Safety and Toxicity in Cynomolgus Monkeys.

To assess safety and toxicity of anti-CD22 ADCs in a primate model,thirty cyno monkeys were assigned to the following treatment groups:vehicle control (6 animals), hu10F4v3-SMCC-DM1 at doses of 2, 4, and 6mg/m̂2 drug dose (equivalent to 0, 10, 20, and 30 mg/kg antibody dose; 4animals per dosage group), and hu10F4v3-MC-MMAF at doses of 2, 4, and 6mg/m̂2 (4 animals per dosage group). Animals were dosed intravenously onDay 1 and Day 22. The animals were evaluated for changes in body weight,food consumption, and pathology indices. Blood samples were collectedand assayed to assess toxicological, pharmacodynamic, and anti-drugantibody effects. One-half of the animals in each group were euthanizedat each of Day 25 and Day 43 and tissue samples were collected.

No noticeable body weight changes were noted in either ADC group. Levelsof serum liver enzymes AST (aspartate aminotransferase), ALT(aminotransferase) and GGT (gamma-glutamyltranspeptidase) were assayedaccording to standard methods well known in the relevant arts.Reversible increases in serum liver enzymes were observed in animalsdosed at 30 mg/kg with either ADC, although ALT was elevated in the DM1group, whereas AST and GGT were elevated in the MMAF group. Sciaticnerve degeneration was minimal to mild in the DM1 group in 2 of 4animals at a dose of 20 mg/kg and in 4 of 4 animals at a dose of 30mg/kg. Sciatic nerve degeneration was minimal in the MMAF group in 1 of4 animals at a dose of 30 mg/kg. Tissue from various organs was examinedmicroscopically. Two of four animals in the 30 mg/kg MMAF group had lunglesions of unknown significance, whereas none were observed in the DM1group.

Depletion of peripheral B cells by the hu10F4v3-MC-MMAF and -SMCC-DM1ADCs was determined by measuring CD20⁺ cell levels in blood over 43 daysin cyno monkeys dosed at Day 0 and Day 22. Blood collected periodicallyduring the study was assayed by FACS using a fluorescently labeledanti-CD20 antibody. The anti-CD22 MMAF and DM1 ADCs deplete cynoperipheral B cells as shown in FIG. 22A (MMAF group) and FIG. 22B (DM1group). No significant effects of MMAF or DM1 ADCs were observed forother lymphocyte populations as shown in FIGS. 23A and 23B in which itis shown that CD4⁺ cells were not significantly depleted over the sametime period.

Hu10F4v3-SMCC-DM1 depleted germinal center B cells in the cyno monkeytonsil samples relative to control as shown in the photomicrographs inFIGS. 24A and 24B. Exemplary germinal centers are circled in FIG. 24A.Complete ablation of germinal center B cells was observed at the 10mg/kg dose level as shown if FIG. 24B. The same results were obtainedfollowing administration of the hu10F4v3-MC-MMAF ADC under the sameconditions.

Hu10F4v3-MC-MMAF dosed at 10 mg/kg depleted dividing B cells from thespleen follicle germinal centers of cyno monkeys. See the diagram inFIG. 25A and the tissue photomicrographs in FIGS. 25B and 25C. The sameresults were obtained when the hu10F4v3-SMCC-DM1 ADC was tested underthe same conditions. Germinal centers appear as dark regions in FIG. 25Busing Ki-67 stain and as unstained areas surrounded by dark regions whenstained with detectably labeled anti-IgD in FIG. 25D. Loss of thegerminal centers due to depletion of germinal center B cells byanti-10F4v3-MC-MMAF is shown in FIGS. 25C and 25E. Thus, theseanti-mitotic drugs have an impact on proliferating B cell populations.

The following hybridoma has been deposited with the American TypeCulture Collection, PO Box 1549, Manassas, Va., 20108, USA (ATCC):

Cell Lines ATCC Accession No. Deposit Date Hybridoma 10F4.4.1 PTA-7621May 26, 2006 Hybridoma 5E8.1.8 PTA-7620 May 26, 2006

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable deposit for 30 years fromthe date of deposit. These cell lines will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the cell lines to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the cell lines to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the depositedcell lines should be lost or destroyed when cultivated under suitableconditions, they will be promptly replaced on notification with aspecimen of the same cell line. Availability of the deposited cell linesis not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literatures cited herein are expressly incorporated in theirentirety by reference.

1-238. (canceled)
 239. A method of treating a B cell proliferativedisorder comprising administering to an individual an effective amountof a pharmaceutical composition comprising an antibody that binds toCD22, comprising: (a) an HVR-L1 of SEQ ID NO: 10; (b) an HVR-L2 of SEQID NO: 12; (c) an HVR-L3 of SEQ ID NO: 14; (d) an HVR-H1 of SEQ ID NO:2; (e) an HVR-H2 of SEQ ID NO: 4; and (f) an HVR-H3 of SEQ ID NO: 6; oran immunoconjugate thereof, having the formula Ab-(L-D)p, wherein Ab issaid anti-CD22 antibody, L is a linker and D is a drug moiety; andwherein said antibody is covalently attached to a cytotoxic agent andwherein the cytotoxic agent is selected from a toxin, a therapeuticagent, a drug moiety, an antibiotic, a radioactive isotope, and anucleolytic enzyme, and a pharmaceutically acceptable carrier.
 240. Amethod of claim 239, wherein the B cell proliferative disorder isselected from lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL,relapsed aggressive NHL, relapsed indolent NHL, relapsed NHL, refractoryNHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acutelymphocytic leukemia (ALL), and mantle cell lymphoma.
 241. A method ofclaim 240, wherein the aggressive NHL is diffuse large B-cell lymphoma(DLBCL).
 242. A method of claim 240, wherein the B cell proliferativedisorder is relapsed NHL or relapsed NHL.
 243. A method of inhibiting Bcell proliferation comprising exposing a cell to an antibody that bindsto CD22 and comprises (i) an HVR-L1 of SEQ ID NO: 10; (ii) an HVR-L2 ofSEQ ID NO: 12; (iii) an HVR-L3 of SEQ ID NO: 14; (iv) an HVR-H1 of SEQID NO: 2; (v) an HVR-H2 of SEQ ID NO: 4; and (vi) an HVR-H3 of SEQ IDNO: 6; or an immunoconjugate thereof, having the formula Ab-(L-D)p,wherein Ab is said anti-CD22 antibody, L is a linker and D is a drugmoiety; and wherein said antibody is covalently attached to a cytotoxicagent and wherein the cytotoxic agent is selected from a toxin, atherapeutic agent, a drug moiety, an antibiotic, a radioactive isotope,and a nucleolytic enzyme.
 244. The method of claim 243, wherein the Bcell proliferative disorder is selected from lymphoma, non-Hodgkinslymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsedindolent NHL, relapsed NHL, refractory NHL, refractory indolent NHL,chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma,leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL),and mantle cell lymphoma.
 245. A method of claim 244, wherein theaggressive NHL is diffuse large B-cell lymphoma (DLBCL).
 246. A methodof claim 244, wherein the B cell proliferative disorder is relapsed NHLor relapsed NHL.
 247. The method of claim 243, wherein the B cell is axenograft.
 248. The method of claim 243, wherein the exposing takesplace in vitro.
 249. The method of claim 243, wherein the exposing takesplace in vivo.
 250. A method of inhibiting cellular proliferationcomprising treating mammalian cancerous B cells in a cell culture mediumwith an antibody that binds to CD22 and comprises (i) an HVR-L1 of SEQID NO: 10; (ii) an HVR-L2 of SEQ ID NO: 12; (iii) an HVR-L3 of SEQ IDNO: 14; (iv) an HVR-H1 of SEQ ID NO: 2; (v) an HVR-H2 of SEQ ID NO: 4;and (vi) an HVR-H3 of SEQ ID NO: 6; or an immunoconjugate thereof,having the formula Ab-(L-D)p, wherein Ab is said anti-CD22 antibody, Lis a linker and D is a drug moiety; and wherein said antibody iscovalently attached to a cytotoxic agent and wherein the cytotoxic agentis selected from a toxin, a therapeutic agent, a drug moiety, anantibiotic, a radioactive isotope, and a nucleolytic enzyme, wherebyproliferation of the cancerous B cells is inhibited.
 251. A method oftreating cancer comprising administering to a patient a pharmaceuticalformulation comprising an antibody that binds to CD22 and comprises (i)an HVR-L1 of SEQ ID NO: 10; (ii) an HVR-L2 of SEQ ID NO: 12; (iii) anHVR-L3 of SEQ ID NO: 14; (iv) an HVR-H1 of SEQ ID NO: 2; (v) an HVR-H2of SEQ ID NO: 4; and (vi) an HVR-H3 of SEQ ID NO: 6; or animmunoconjugate thereof, having the formula Ab-(L-D)p, wherein Ab issaid anti-CD22 antibody, L is a linker and D is a drug moiety; andwherein said antibody is covalently attached to a cytotoxic agent andwherein the cytotoxic agent is selected from a toxin, a therapeuticagent, a drug moiety, an antibiotic, a radioactive isotope, and anucleolytic enzyme, and a pharmaceutically acceptable diluent, carrieror excipient.
 252. The method of claim 251 wherein the cancer isselected from the group consisting of wherein the B cell proliferativedisorder is selected from lymphoma, non-Hodgkins lymphoma (NHL),aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, relapsedNHL, refractory NHL, refractory indolent NHL, chronic lymphocyticleukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cellleukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.
 253. A method of claim 252, wherein the aggressive NHL isdiffuse large B-cell lymphoma (DLBCL).
 254. A method of claim 252,wherein the B cell proliferative disorder is relapsed NHL or relapsedNHL.
 255. The method of claim 251 wherein the patient is administered acytotoxic agent in combination with the immunoconjugate.
 256. The methodof claim 251 wherein the patient is administered said antibody orimmunoconjugate thereof in combination with an anti-CD20 antibody.